Antimicrobial agents and methods

Abstract

The present invention relates to means for performing conjugation between bacteria, and in particular, the present invention relates to a carrier bacterium comprising an antimicrobial agent and methods of use. The carrier bacterium is capable of transferring DNA encoding the agent to a target cell by conjugation. The present invention further relates to promoting growth or feed conversion in an animal. The present invention further relates to killing Salmonella or inhibiting growth or proliferation of Salmonella.

Description

Technical Field

[0001] This invention relates to means for conjugating bacteria, and more particularly, to carrier bacteria containing antimicrobial agents and methods of use. The carrier bacteria are capable of conjugating and transferring DNA encoding the reagent to target cells.

[0002] This invention further relates to promoting animal growth or feed conversion ratio (FCR). This invention further relates to killing or inhibiting the growth or proliferation of Salmonella spp. This invention further relates to killing Pseudomonas spp. Pseudomonas It can also inhibit the growth or proliferation of Pseudomonas species, for example, it can be used to promote plant growth, dry weight, wet weight or crop production. Background Technology

[0003] The DNA sequences controlling extrachromosomal replication (ori) and transfer (tra) are distinct; that is, replication sequences generally do not control plasmid transfer, or vice versa. Replication and transfer are two complex molecular processes utilizing both plasmid and host coding functions. Bacterial conjugation is the unidirectional and horizontal transfer of genetic information from one bacterium to another. The transferred genetic material can be a plasmid, or it can be a part of a chromosome. Bacterial cells with conjugation plasmids contain surface structures (sex pili) involved in the coupling of donor and recipient cells and the transfer of genetic information. Conjugation involves cell-to-cell contact, and the transfer of genetic traits can be mediated by many plasmids. Of all natural transfer mechanisms, conjugation is the most efficient. For example, the F plasmid of *Escherichia coli*, *Enterococcus faecalis*, and *Enterococcus faecium*... Enterococcus faecalis pCFlO plasmid and Bacillus thuringiensis ( Bacillus thuringiensis The pXO16 plasmid employs different mechanisms for establishing mating pairs, resulting in mating aggregates of varying sizes and different host ranges within Gram-negative (F) and Gram-positive bacteria (pCF10 and pXO16). Their plasmid sizes also differ; they are 54, 100, and 200 kb, respectively. However, it is noteworthy that these conjugation systems share a crucial common characteristic: they support conjugation transfer in liquid media and often achieve near-100% transfer efficiency within a short timeframe. Therefore, the conjugation process allows for the protection of plasmid DNA from environmental nucleases and the very efficient delivery of plasmid DNA into recipient cells. The conjugation function is encoded by the native plasmid. Numerous conjugation plasmids (and transposons) are known to transfer related genes within a single species (narrow host range) or between many species (wide host range). Transferable plasmids have been reported in numerous Gram-positive genera, including but not limited to Streptococcus. Streptococcus Staphylococcus spp. Staphylococcus ), Bacillus spp. BacillusClostridium ( Clostridium ) and Nocardia spp. Nocardia The pathogenic strains of Gram-negative bacteria. The early stages of conjugation generally differ in Gram-negative and Gram-positive bacteria. Some transfer genes in conjugation plasmids from Gram-negative bacteria serve to provide pili-mediated intercellular contact, conjugation pore formation, and related morphological functions. Pili do not appear to be involved in initiating conjugation in Gram-positive bacteria. Summary of the Invention

[0004] This invention provides:

[0005] A method for enhancing the growth or weight of a subject (e.g., non-medical or medical), wherein the subject comprises bacterial target cells, the method comprising administering to the subject a first additional DNA encoding an antimicrobial agent that is toxic to the target cells, wherein the first DNA is transferred into the target cells and the reagent is expressed, thereby killing the target cells in the subject or reducing the growth or proliferation of the target cells, and enhancing the growth or weight of the subject.

[0006] In one implementation, the following is provided:

[0007] A method for enhancing the growth or weight of a subject (e.g., non-medical or medical), wherein the method comprises administering to the subject multiple carrier cells, wherein the subject comprises bacterial target cells, and each carrier cell is a bacterial cell containing a first additional DNA encoding an antimicrobial agent that is toxic to the target cells but non-toxic to the carrier cells, the carrier cells being capable of DNA binding transfer into the target cells for expression of the agent therein, wherein the first DNA is transferred from the carrier cells into the target cells for expression therein to produce the antimicrobial agent, thereby killing or reducing the growth or proliferation of the target cells in the subject, and enhancing the growth or weight of the subject.

[0008] Advantageously, the FCR in the subjects is improved (i.e., enhanced), that is, the FCR number is reduced. When the method of the invention is performed on a group of subjects (e.g., a group of animals, such as livestock), the FCR is reduced in individuals within that group or the average FCR is reduced within that group. The reduction can be evaluated as a comparison with the FCR before the administration of the carrier cells, or with the average of animals of the same species, age group, and fed with a comparable or identical diet. A skilled person will be familiar with the standard FCR of, for example, livestock such as piglets, pigs, sheep, cattle (dairy or beef cattle), fish, shellfish, and poultry (e.g., chickens (broilers or laying hens), geese, ducks, or turkeys).

[0009] The present invention also provides:

[0010] A carrier cell, wherein the cell is a bacterial cell comprising a first episomal DNA encoding an antibacterial agent that is toxic to a bacterial target cell but not to the carrier cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression therein of the antibacterial agent to kill the target cell, wherein the target cell is a Salmonella cell and the carrier cell is an Enterobacteriaceae cell.

[0011] A composition comprising a plurality of carrier cells for use in a method comprising administering the cells to a subject to treat an infection of pathogenic bacterial target cells, wherein each carrier cell is a bacterial cell comprising a first episomal DNA encoding an antibacterial agent that is toxic to the target cell but not to the carrier cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression therein of the agent, wherein the first DNA is transferred from the carrier cell into the target cell for expression therein to produce the antibacterial agent to kill or reduce growth or proliferation of the target cells in the subject, wherein the target cell is a Salmonella cell and the carrier cell is an Enterobacteriaceae cell.

[0012] A non-medical method of killing animal-derived bacterial target cells in an animal, optionally a livestock animal, the method comprising administering to the animal a plurality of carrier cells, wherein each carrier cell is a bacterial cell comprising a first episomal DNA encoding an antibacterial agent that is toxic to the target cell but not to the carrier cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression therein of the agent, wherein the first DNA is transferred from the carrier cell into the target cell for expression therein to produce the antibacterial agent to kill or reduce growth or proliferation of the target cells in the subject, wherein the target cell is a Salmonella cell and optionally the carrier cell is an Enterobacteriaceae cell.

[0013] DNA, optionally for use in a method of the invention, wherein the DNA is capable of being introduced into a target cell, wherein the DNA encodes a plurality of guide RNAs or crRNAs of a CRISPR / Cas system, wherein the guide RNAs or crRNAs are operable with a Cas nuclease in the target cell to recognize a plurality of protospacer sequences comprised by a genome of the target cell; wherein

[0014] (a) the protospacer sequences comprise one or more pathogenicity island nucleotide sequences of the genome of the target cell;

[0015] (b) the protospacer sequences comprise one or more invasin gene sequences of the genome of the target cell;

[0016] (c) the protospacer sequences comprise one or more secretion system gene sequences of the genome of the target cell; and / or

[0017] (d) the pro-interval sequence comprises one or more nucleotide sequences selected from the following genes of Salmonella, and orthologues or homologues thereof: avrA, sptP, sicP, sipA, sipD, sipC, sipB, sicA, invB, sseE, sseA, sseB, sscA, sseC, sseD, sseE, sscB, sseF, sseG, mgtC, cigR, pipA, pipB, pipC, sopB and pipD optionally selected from invB, sicP, sseE, pipA, pipB, pipC, hilA, marT and sopB ).

[0018] The application also provides the following configurations:

[0019] In a first configuration

[0020] A carrier bacterial cell comprising first episomal DNA, the DNA encoding a nucleic acid sequence of interest (NSI) or encoding an antibacterial agent, the antibacterial agent being toxic to a target bacterial cell but not to the carrier cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression of the agent therein, optionally wherein

[0021] (a) the carrier cell comprises second DNA distinct from the first DNA, wherein the second DNA comprises or encodes a first factor required for replication of the first DNA;

[0022] (b) the first DNA does not comprise or encode the first factor, wherein the first DNA is non-self-replicating in the absence of the first factor but is capable of replication in the carrier cell in the presence of the first factor provided by the second DNA;

[0023] (c) wherein the cell comprises a gene encoding one or more conjugation factors sufficient to effect conjugative transfer of the first DNA into the target bacterial cell.

[0024] In embodiments, the application effectively recognises the benefit of using an antibacterial agent that acts by target sequence recognition in the target cell genome rather than in the carrier cell, which releases the ability of the first DNA to replicate freely in the carrier cell without toxicity to the carrier cell.

[0025] In a second configuration

[0026] DNA encoding a NSI or encoding an antibacterial agent, the antibacterial agent being toxic to a target bacterial cell but not to the carrier cell, the carrier cell being capable of carrying DNA for conjugative transfer into the target cell, wherein the first DNA does not comprise or encode a first factor required for replication of the first DNA, and wherein the first DNA lacks a component required for conjugative transfer of the first DNA into the target bacterial cell.

[0027] In a third configuration

[0028] A method for enhancing growth or weight of a human or animal subject, wherein the method comprises administering a plurality of the vector cells according to the application to the microbiota of the subject, wherein the microbiota comprises target cells, and the first DNA is transferred from the vector cells into the target cells for expression therein to produce an antibacterial agent, thereby killing or reducing growth or proliferation of the target cells (e.g. Salmonella cells) in the subject.

[0029] In a fourth configuration

[0030] A method for enhancing growth or weight of a plant, wherein the method comprises administering a plurality of the vector cells according to the application to the microbiota of the plant, wherein the microbiota comprises target cells, and the first DNA is transferred from the vector cells into the target cells for expression therein to produce an antibacterial agent, thereby killing or reducing growth or proliferation of the target cells (e.g. Pseudomonas cells) in the plant.

[0031] In a fifth configuration

[0032] A method for reducing a biofilm comprised by or on a surface of a subject, wherein the biofilm comprises target cells (e.g. Pseudomonas cells), wherein the method comprises administering a plurality of the vector cells according to the application to the biofilm, wherein the first DNA is transferred from the vector cells into the target cells for expression therein to produce an antibacterial agent, thereby killing or reducing growth or proliferation of the target cells in the biofilm.

[0033] In a sixth configuration

[0034] A method of replicating a first DNA (e.g. comprised by a conjugative plasmid) to produce a plurality of copies of the DNA, the method comprising culturing a plurality of the vector bacterial cells according to the application, wherein the first DNA is replicated in the cells; and optionally isolating the plurality of copies of the first DNA from the vector cells.

[0035] In a seventh configuration

[0036] A method of killing or reducing growth or proliferation of a plurality of target bacterial cells, the method comprising

[0037] (a) obtaining a sample of the vector cells according to the application or obtainable by the method of the sixth configuration;

[0038] (b) contacting the sample of vector cells with a plurality of target bacterial cells to allow conjugation between the vector cells and the target cells; and

[0039] (c) allowing copies of the first DNA to be transferred from the vector cells to the target cells by conjugation, wherein the antibacterial agent is provided in the target cells, and the target cells are killed or their growth or proliferation is reduced;

[0040] (d) wherein the first DNA is not (or is not substantially) replicable in the target cell.

[0041] In an eighth configuration

[0042] A pharmaceutical composition, livestock growth promoting composition, zoonosis control agent, biocide for administration to livestock, soil amendment, herbicide, plant fertilizer, food or food ingredient sterilization composition, dental composition, personal hygiene composition, or disinfectant composition (e.g., for domestic or industrial use) comprising a plurality of the vector cells according to the application.

[0043] In a ninth configuration

[0044] In a first aspect:

[0045] A method of promoting growth of an animal (e.g., a livestock animal, e.g., a poultry animal), the method comprising administering to the animal a guide nuclease system or component thereof, and introducing the system or component into a target bacterium comprised by the animal, wherein the guide nuclease is capable of recognizing and modifying (e.g., cleaving) a target nucleotide sequence comprised by the target bacterium, thereby killing the target bacterium or inhibiting growth or proliferation of the target bacterium, and promoting growth of the animal.

[0046] The method is a non-medical method, and the target bacterium present in the animal is capable of inhibiting growth of the animal. Thus, the method reduces the burden of such bacteria in the animal and promotes growth.

[0047] In a second aspect:

[0048] A method of enhancing feed conversion ratio (FCR) in an animal (e.g., a livestock animal, e.g., a poultry animal), the method comprising administering to the animal a guide nuclease system or component thereof, and introducing the system or component into a target bacterium comprised by the animal, wherein the guide nuclease is capable of recognizing and modifying (e.g., cleaving) a target nucleotide sequence comprised by the target bacterium, thereby killing the target bacterium or inhibiting growth or proliferation of the target bacterium, and increasing FCR of the animal.

[0049] The method is a non-medical method, and the target bacterium present in the animal is capable of increasing FCR of the animal. Thus, the method reduces the burden of such bacteria in the animal and enhances FCR (i.e., reduces the number of FCR).

[0050] In a third aspect:

[0051] A method of promoting growth of an animal (e.g., a livestock animal, e.g., a poultry animal), the method comprising administering to the animal an antibacterial agent that is toxic to Salmonella bacteria, wherein Salmonella target bacteria comprised by the animal are exposed to the agent and killed, or growth or proliferation of the target bacteria is inhibited, and growth of the animal is promoted.

[0052] The method is a non-medical method and the target bacteria present in the animal are capable of inhibiting the growth of the animal. Thus, the method reduces the burden of such bacteria in the animal and promotes growth.

[0053] In a fourth aspect:

[0054] A method of enhancing feed conversion ratio (FCR) in an animal (e.g. a livestock animal, e.g. a poultry animal), the method comprising administering to the animal an antibacterial agent that is toxic to Salmonella bacteria, wherein target Salmonella bacteria comprised by the animal are exposed to the agent and killed, or the growth or proliferation of the target bacteria is inhibited, and the FCR of the animal is enhanced.

[0055] The method is a non-medical method and the target bacteria present in the animal are capable of increasing the FCR of the animal. Thus, the method reduces the burden of such bacteria in the animal and enhances the FCR (i.e. reduces the FCR number).

[0056] In a tenth configuration

[0057] In a first aspect:

[0058] A method of promoting growth, dry weight, wet weight or crop production of a plant, the plant comprising a microbiota comprising target bacteria, the method comprising contacting the microbiota with an antibacterial agent that is toxic to the target bacteria, wherein the target bacteria are killed or the growth or proliferation of the target bacteria is inhibited, and the growth, dry weight, wet weight or crop production of the plant is increased.

[0059] In a second aspect:

[0060] Use of an antibacterial agent that is toxic to target bacteria in a method for promoting growth, dry weight, wet weight or crop production of a plant, in which method target bacteria comprised by a microbiota of the plant are contacted with the agent, whereby the target bacteria are killed or the growth or proliferation of the target bacteria is inhibited.

[0061] In a third aspect:

[0062] A method of increasing crop yield of a plant, the plant comprising a microbiota comprising target bacteria, the method comprising contacting the microbiota with an antibacterial agent that is toxic to the target bacteria, wherein the target bacteria are killed or the growth or proliferation of the target bacteria is inhibited, and the growth, dry weight, wet weight or crop production of the plant is increased.

[0063] In a fourth aspect:

[0064] Use of an antibacterial agent that is toxic to target bacteria in a method for increasing crop yield of a plant, in which method target bacteria comprised by a microbiota of the plant are contacted with the agent, whereby the target bacteria are killed or the growth or proliferation of the target bacteria is inhibited.

[0065] In a fifth aspect:

[0066] A method of increasing leaf chlorophyll of a plant comprising a microbiota comprising target bacteria, the method comprising contacting the microbiota with an antibacterial agent toxic to the target bacteria, wherein the target bacteria are killed or inhibited from growing or proliferating, and the leaf chlorophyll of the plant is increased.

[0067] In a sixth aspect:

[0068] Use of an antibacterial agent toxic to target bacteria in a method for increasing leaf chlorophyll of a plant, in which the target bacteria comprised by a microbiota of the plant are contacted with the agent, whereby the target bacteria are killed or inhibited from growing or proliferating.

[0069] In a seventh aspect:

[0070] A method of increasing greening of a plant comprising a microbiota comprising target bacteria, the method comprising contacting the microbiota with an antibacterial agent toxic to the target bacteria, wherein the target bacteria are killed or inhibited from growing or proliferating, and the greening of the plant is increased.

[0071] In an eighth aspect:

[0072] Use of an antibacterial agent toxic to target bacteria in a method for increasing greening of a plant, in which the target bacteria comprised by a microbiota of the plant are contacted with the agent, whereby the target bacteria are killed or inhibited from growing or proliferating.

[0073] In a ninth aspect:

[0074] A method of reducing a biofilm (e.g., a leaf biofilm) comprised by a plant comprising a microbiota comprising target bacteria, the method comprising contacting the microbiota with an antibacterial agent toxic to the target bacteria, wherein the target bacteria are killed or inhibited from growing or proliferating, and the biofilm of the plant is reduced.

[0075] In a tenth aspect:

[0076] Use of an antibacterial agent toxic to target bacteria in a method for inhibiting a biofilm of a plant, in which the target bacteria comprised by a biofilm (e.g., a leaf biofilm) of the plant are contacted with the agent, whereby the target bacteria are killed or inhibited from growing or proliferating.

[0077] Optionally, the target bacteria are Pseudomonas bacteria, e.g., Pseudomonas syringae P syringae ) or Pseudomonas aeruginosa P aeruginosa ) bacteria or any other Pseudomonas bacteria disclosed herein.

[0078] Optionally, the agent is a guided nuclease system or a component thereof, e.g., any such system or component disclosed herein for modifying (e.g., cleaving) a target nucleic acid sequence comprised by the target bacteria.

[0079] Optionally, the plant is any plant disclosed herein.

[0080] Optionally, the chlorophyll is chlorophyll a and / or chlorophyll b.

[0081] Optionally, the agent is comprised by a carrier cell of the invention, and the contacting comprises contacting the cell with the carrier cell. BRIEF DESCRIPTION OF DRAWINGS

[0082] Figure 1 . PCR performed on recipient colonies. From left to right: 1) Plasmid pFSMobC* (control). 2) Plasmid pFSMobC* in S17 (control). 3) Colony from transconjugant plate (Cm30+ Na125). 4) Colony n°2 from transconjugant plate (Cm30+ Na125);

[0083] Figure 2 : Effect of pFSMobSal7 delivered by conjugation on the viability of Salmonella enteritidis FS26;

[0084] Figure 3 . Number of Salmonella in cecum 7 days post-infection. N=15 birds / group;

[0085] Figure 4 . Frequency of detection of Salmonella in cecum 7 days post-infection. N=15 birds / group; and

[0086] Figure 5 . Post-mortem carcass weight of birds at 6 weeks of age.

[0087] Figure 6 . Number of Salmonella in crop digesta (CFU / g) 7 days post-challenge; N=9 birds; P=0.03. DETAILED DESCRIPTION

[0088] The present invention relates to means for performing conjugation between bacteria, and in particular, the present invention relates to carrier bacteria comprising an antimicrobial agent and methods of use. The carrier bacteria are capable of conjugative transfer of DNA encoding an agent to a target cell.

[0089] The present invention further relates to promoting growth, weight, or feed conversion ratio (FCR) in an animal. The present invention further relates to killing or inhibiting growth or proliferation of Salmonella.

[0090] Thus, there is provided:

[0091] A method (e.g., non-medical or medical) for enhancing growth or weight in a subject, wherein the subject comprises bacterial target cells, the method comprising administering to the subject a first episomal DNA encoding an antibacterial agent, the antibacterial agent being toxic to the target cells, wherein the first DNA is transferred into the target cells and the agent is expressed, thereby killing or reducing growth or proliferation of the target cells in the subject, and enhancing growth or weight in the subject.

[0092] Advantageously, the FCR is enhanced in the subject. Optionally, the FCR is enhanced (i.e., reduced) by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%, e.g., from 2 to 6% or from 2 to 5% (e.g., 2, 3, 4, 5, or 6%) compared to the FCR of the subject prior to administration of the agent or the carrier cell. Optionally, the FCR is enhanced by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%, e.g., 2-5% (e.g., 2, 3, 4, or 5%) compared to the FCR of a control subject of the same species and gender that has not received administration of the agent or the carrier cell. In one embodiment, the subject is a bird, an avian bird, a chicken, a turkey, a goose, or a duck (preferably a chicken), and the FCR number is reduced by an amount of 0.03 to 0.07, e.g., 0.04 to 0.06, e.g., 0.04, 0.05, or 0.06. Thus, for example, the FCR of a control bird is 1.7 (g feed / g poultry) compared to a control bird, and after the subject bird has been treated using the present invention, the subject bird has an FCR of 1.64 to 1.66. The control bird (e.g., chicken) is of the same species, gender, and age as the subject of the present invention, and is fed the same diet (feed), but has not received treatment of the present invention. The birds can be from the same flock. Thus, in one embodiment, the bird (e.g., chicken) has an FCR of 1.64 to 1.66, wherein the subject has been treated by the method of the present invention.

[0093] In one example, the method is performed on a group of subjects (e.g. subjects of the same species, e.g. a group of livestock animals, e.g. a flock of poultry or a herd of cattle or a herd of sheep). In this example, the average FCR of the group is enhanced (i.e. the FCR number is reduced) by the method compared to a control group of animals of the same species, subspecies or type, or compared to the average FCR of the group prior to treatment with the method. The control group can be a group of animals of the same species having the same average age, the same ratio of males to females, and fed on the same diet as the group of the application. For example, both the group of the application and the control group are flocks of poultry (e.g. chickens), e.g. having a common ancestor one or two or three generations back. The flock can be a broiler flock or a layer flock. For example, both the group of the application and the control group are herds of beef cattle or dairy cattle, e.g. having a common ancestor one or two or three generations back.

[0094] FCR, FE and ECI

[0095] In livestock farming, feed conversion ratio (FCR) is a ratio that measures the efficiency of an animal's body to convert animal feed into a desired output. For example, for dairy cattle, the output is milk, while in animals raised for meat, such as beef cattle, pigs, chickens and fish, the output is meat, i.e. the increase in body mass by the animal, expressed in the final mass of the animal or the mass of the dressed output. FCR is the mass of input divided by the mass of output (thus mass of feed / mass of milk or meat). In some fields, feed efficiency (FE) is used, which is the inverse of FCR, i.e. output divided by input. These concepts are also closely related to the efficiency of conversion of ingested food (ECI). FCR is widely used in pig and poultry production, while FE is more commonly used for cattle. As a ratio, FCR is dimensionless, i.e. it is not affected by the units of measurement used to determine FCR. Animals with a low FCR are considered to be efficient users of feed.

[0096] Preferably, the enhancement produced by the application is an increase in body mass of the subject. Instead of an enhancement of FCR, the application can be a method for enhancing feed efficiency (FE) in a subject or enhancing efficiency of conversion of ingested food (ECI) in a subject.

[0097] FCR can be calculated using dry mass of feed, or can be calculated based on wet mass at feeding.

[0098] In one embodiment, the FCR, FE or ECI number is changed by 0.2, 0.5, 1, 1.5 or 2. For example, the FCR is reduced by 1, 1.5 or 2.

[0099] Conversion of livestock

[0100] Beef cattle

[0101] As of 2013, in the United States, FCRs calculated as 4.5 - 7.5 based on live weight are in the normal range, with FCRs higher than 6 being typical. Thus, in one example of the invention, the animal or each animal is a beef cattle, and the number of FCRs in the animal (or the average FCR of the animals) is reduced by the method to less than 6 (calculated using the live weight of the animal), for example, less than 5.5, 5, 4.5, 4, or 3.5 (optionally, the FCR or average FCR is reduced to a FCR of 3.5 to 5.9, for example, 4 to 5.5.

[0102] Dairy cattle

[0103] In the United States, the price of milk is based on protein and fat content, so FCRs are often calculated taking this into account. Using FCRs calculated based only on the weight of protein and fat in the milk obtained from the animal, as of 2011, FCRs of 13 are very poor, while FCRs of 8 are very good. Thus, in one example of the invention, the animal or each animal is a dairy cattle, and the number of FCRs in the animal (or the average FCR of the animals) is reduced by the method to less than 11, 10, 9, or 8, (and optionally greater than 5 or 6), where the FCR is the FCR of the milk obtained from the animal that has been treated by the methods of the invention, calculated using the combined weight of protein and fat in the milk.

[0104] Another method used for handling pricing based on protein and fat is to use energy-corrected milk (ECM), which assumes a certain amount of fat and protein in the final dairy product, adding a factor of standardization; the formula is (0.327 x milk mass) + (12.95 x fat mass) + (7.2 x protein mass). In the dairy industry, feed efficiency (ECM / intake) is often used instead of FCR (intake / ECM); FE less than 1.3 is considered problematic. Thus, in one example of the invention, the animal or each animal is a dairy cattle, and the number of FE in the animal (or the average FE of the animals) is increased by the method to more than 1.3, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, or 3, (and optionally not more than 4 or 5). Optionally, the FE is increased to more than 1.3, 1.5, 1.6, and not more than 1.7.

[0105] FE based on milk weight only is also used; FE of 1.30 to 1.70 is normal. Thus, in one example of the invention, the animal or each animal is a dairy cattle, and the number of FE in the animal (or the average FE of the animals) is increased by the method to more than 1.3, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, or 3, (and optionally not more than 4 or 5). Optionally, the FE is increased to more than 1.3, 1.5, 1.6, and not more than 1.7.

[0106] pig

[0107] As at 2011, pigs used commercially in the UK and Europe have an FCR (calculated using weight gain) of about 1 as piglets, and terminate at slaughter at about 3. As at 2012, in Australia and using dressed weight for output, FCRs calculated using dressed carcass weight are fair at 4.5, good at 4.0, and excellent at 3.8. In the US, as at 2012, commercial pigs have FCRs calculated using weight gain, which are 3.46 when they weigh 240 to 250 pounds, 3.65 when they weigh 250 to 260 pounds, 3.87 when they weigh 260 to 270 pounds, and 4.09 when they weigh 280 to 270 pounds. Thus, in one example of the application, the animal or each animal is a piglet, and the number of FCRs in the animal (or the average FCR of the animals) is reduced to 1 or less by the method, the FCRs being calculated using weight gain of the animal. Thus, in one example of the application, the animal or each animal is a pig of up to 3 months of age, and the number of FCRs in the animal (or the average FCR of the animals) is reduced to 1 or less by the method, the FCRs being calculated using weight gain of the animal. Thus, in one example of the application, the animal or each animal is a pig of greater than 3 months of age but up to 6 or 7 months of age, and the number of FCRs in the animal (or the average FCR of the animals) is reduced to 3 or less (e.g. from 1 to 3) by the method, the FCRs being calculated using weight gain of the animal. The piglet can be 1.5 to 3 months of age; the pig for slaughter can be greater than 3 months of age but up to 6 months of age. Thus, in one example of the application, the animal or each animal is a pig of 240 to 250 pounds in weight, and the number of FCRs in the animal (or the average FCR of the animals) is reduced to 3.5 or less (e.g. 3.4 or less, but optionally not less than 3 or 2.5) by the method, the FCRs being calculated using weight gain of the animal. Thus, in one example of the application, the animal or each animal is a pig of 250 to 260 pounds in weight, and the number of FCRs in the animal (or the average FCR of the animals) is reduced to 3.7 or less (e.g. 3.65 or less, but optionally not less than 3 or 3.5) by the method, the FCRs being calculated using weight gain of the animal. Thus, in one example of the application, the animal or each animal is a pig of 260 to 270 pounds in weight, and the number of FCRs in the animal (or the average FCR of the animals) is reduced to 3.9 or less (e.g. 3.8 or less, but optionally not less than 3.7 or 3.5) by the method, the FCRs being calculated using weight gain of the animal. Thus, in one example of the application, the animal or each animal is a pig of 280 to 270 pounds in weight, and the number of FCRs in the animal (or the average FCR of the animals) is reduced to 4.1 or less (e.g. 4 or less, but optionally not less than 3.8 or 3.9) by the method, the FCRs being calculated using weight gain of the animal.

[0108] Sheep

[0109] Some data on sheep illustrates the variation in FCR. FCR (kg feed dry matter intake / kg live weight gain) for lambs often ranges from about 4 to 6. Thus, in one example of the invention, the animal or each animal is a sheep, and the number of FCRs in the animal (or the average FCR for the animals) is reduced by the method to 6 or less (e.g., from 4 to 6), the FCRs being calculated using kg feed dry matter intake / kg live weight gain of the animal.

[0110] Poultry

[0111] As of 2011, broiler chickens in the United States typically have an FCR of 1.6 based on weight gain, and mature in 39 days. At about the same time, broiler chickens in Brazil had an FCR of 1.8 based on weight gain. Thus, in one example of the invention, the animal or each animal is a poultry bird (e.g., a broiler chicken), and the number of FCRs in the animal (or the average FCR for the animals) is reduced by the method to 1.9, 1.8, 1.7, 1.6 or less (e.g., from 1.9 to 1.5; or from 1.8 to 1.6), the FCRs being calculated based on weight gain. Optionally, the FCR is calculated when each animal is 35-40, e.g., 39, days of age.

[0112] For hens used for egg production in the United States, as of 2011, the FCR was about 2, with each hen laying about 330 eggs / year. Thus, in one example of the invention, the animal or each animal is a poultry bird (e.g., a layer chicken, e.g., a hen), and the number of FCRs in the animal (or the average FCR for the animals) is reduced by the method to 2 or less (e.g., from 2 to 1.5), the FCRs being calculated based on weight gain. Thus, in one example of the invention, the animal or each animal is a poultry bird (e.g., a layer chicken, e.g., a hen), and the number of eggs laid by the animal (or the average number of eggs laid by the animals) is more than 330 eggs / year (or proportionally allocated for different time periods), e.g., more than 340, 350, or 400 eggs / year (or proportionally allocated for time periods).

[0113] Meat fish

[0114] The FIFO ratio (or Fish In - Fish Out ratio) is a conversion ratio applied to aquaculture, where the first number is the mass of captured fish used to feed the farmed fish, and the second number is the mass of resulting farmed fish. The FIFO is a way of expressing the contribution from the use of captured wild fish in aquaculture feed, compared to the amount of farmed fish that is edible. As aquaculture has grown and produced more feed, but with a finite annual supply of fish meal and fish oil, the inclusion rate of fish meal and fish oil in aquaculture feed has shown a consistent decrease over time. Calculations have shown that the overall farmed aquaculture FIFO has decreased from 0.63 in 2000 to 0.33 in 2010 and 0.22 in 2015. In 2015, approximately 4.55 kg of farmed fish was produced for every 1 kg of wild fish captured and used in feed. Fish used in fish meal and fish oil production are not used for human consumption, but with their use as fish meal and fish oil in aquaculture feed, they contribute to global food production. Thus, in one example, when the animal is a fish (e.g., salmon, tilapia, or catfish), the method of the present invention enhances (i.e., increases) the FIFO ratio. For example, the FIFO is increased to 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8, 1, 1.5, or 2 or more. For example, the fish is salmon or catfish, and the FIFO is increased to more than 1, 1.5, or 2 (and optionally not more than 2 or 2.5). For example, the fish is tilapia, and the FIFO is increased to more than 1.5 or 2 (and optionally not more than 2 or 2.5).

[0115] The enhancement in growth or weight can be an enhancement (increase) in milk production or yield, e.g., an average increase in the group of the present invention compared to a control group. The control group can be a group of the same species having the same average age, the same male and female ratio, and fed the same diet as the group of the present invention. For example, the subject here can be a dairy cow.

[0116] The enhancement in growth or weight can be an enhancement (increase) in egg production or yield, e.g., an average increase in the group of the present invention compared to a control group. The control group can be a group of the same species having the same average age, the same male and female ratio, and fed the same diet as the group of the present invention. For example, the subject here can be a laying hen.

[0117] The enhancement in growth or weight can be an enhancement (increase) in meat production or yield, e.g., an average increase in the group of the present invention compared to a control group. The control group can be a group of the same species having the same average age, the same male and female ratio, and fed the same diet as the group of the present invention. For example, the subject here can be a dairy cow, poultry (e.g., chicken), fish, shellfish, sheep, or pig.

[0118] The enhancement in growth or weight can be an enhancement (increase) in fat production or yield, e.g. an average increase in the group of the application compared to a control group. The control group can be a group of animals of the same species, with the same average age, the same male and female ratio, and fed with the same diet, as the group of the application. For example, the subject herein can be a dairy cow, poultry (e.g. chicken), fish (e.g. salmon, tilapia or catfish), shellfish, sheep or pig.

[0119] The enhancement in growth or weight can be an enhancement (increase) in fur or hide production or yield, e.g. an average increase in the group of the application compared to a control group. The control group can be a group of animals of the same species, with the same average age, the same male and female ratio, and fed with the same diet, as the group of the application. For example, the subject herein can be a cow.

[0120] In one example, the subject is a shellfish. The shellfish can be selected from the group consisting of shrimp, prawn, crab, lobster, clam, scallop, oyster, prawn and mussel.

[0121] The subject can be any subject disclosed herein. The subject can be an animal, e.g. a livestock animal, e.g. a bird (e.g. a poultry bird; or a chicken or turkey) or a pig. Alternatively, the subject can be a human, e.g. a human having an eating disorder (e.g. anorexia) or insufficient weight, e.g. wherein the human has a body mass index (BMI) of less than 18.5, 18, 17, 16 or 15. For example, the human has mild anorexia (i.e. the human has a BMI < 17.5), moderate anorexia (i.e. the human has a BMI of 16 to 16.99), severe anorexia (i.e. the human has a BMI of 15 to 15.99) or extreme anorexia (i.e. the human has a BMI < 15).

[0122] In one alternative, for example, the subject is a plant and the target bacteria is a plant pathogen. In one example, the target bacteria is Pseudomonas, e.g. Pseudomonas syringae or Pseudomonas aeruginosa.

[0123] In one alternative, the target cell is an archaeal cell. For example, the target cell is a Methanobacterium cell.

[0124] For example, the target cell is a methanogen cell. For example, the target cell comprises cells of one or more species selected from the group consisting of:

[0125] • Methanobrevibacter smithii (M. smithii) Methanobacterium bryantii )

[0126] • Methanocorpusculum parvum (M. parvum) Methanobacterium formicum )

[0127] • Methanoreflexus taylorii (M. taylorii) Methanobrevibacter arboriphilicus )

[0128] •Methanobrevibacter gottschalkii

[0129] • Rumen methanogenic short bacilli ( Methanobrevibacter ruminantium )

[0130] • Methanobacterium spp. ( Methanobrevibacter smithii )

[0131] • Methanococcus chunghsingensis

[0132] • Methanococcus burtonii

[0133] • Methanococcus aeolicus

[0134] • Methanococcus deltae

[0135] • Methanococcus japonicus ( Methanococcus jannaschii )

[0136] • Methanococcus faecium ( Methanococcus maripaludis )

[0137] • Methanococcus vannamei ( Methanococcus vannielii )

[0138] • Rennet methanogens ( Methanocorpusculum labreanum )

[0139] • Bleuse methanogens ( Methanoculleus bourgensis (Olantangi methanogens) Methanogenium olentangyi ) and Bourgeois methanogens ( Methanogenium bourgense ))

[0140] • Black Sea methanophora ( Methanoculleus marisnigri )

[0141] • Methanoflorens stordalenmirensis

[34]

[0142] • Methanofollis liminatans

[0143] • Carriaza methanogens ( Methanogenium cariaci )

[0144] • Reflux methanogens ( Methanogenium frigidum )

[0145] • Organophile methanogens ( Methanogenium organophilum )

[0146] • Methanogen Wolf ( Methanogenium wolfei )

[0147] • Motile methanogenic bacteria ( Methanomicrobium mobile )

[0148] • Methanophilic bacterium Kanni ( Methanopyrus kandleri )

[0149] • Methanoregula boonei

[0150] • Syntropho methanomassiliicoccus Methanosaeta concilii )

[0151] • Methylo thermaspirillum Methanosaeta thermophila )

[0152] • Methanosaeta acetivorans Methanosarcina acetivorans )

[0153] • Methanosaeta paucivorans Methanosarcina barkeri )

[0154] • Methanosaeta marisnigri Methanosarcina mazei )

[0155] • Methanococcus maripaludis Methanosphaera stadtmanae )

[0156] • Methanohalobium evronense Methanospirillium hungatei )

[0157] • Methanothermobacter defluvii (Methanobacterium defluvii) Methanothermobacter defluvii ) Methanobacterium defluvii • Methanothermobacter thermautotrophicus (Methanobacterium thermoautotrophicum)

[0158] • Methanothermobacter curvus (Methanobacterium curvum) Methanothermobacter thermautotrophicus ) Methanobacterium thermoautotrophicum • Methanothermobacter ferdirigidus (Methanobacterium ferdirigidum)

[0159] • Methanothermobacter woesei (Methanobacterium woesei) Methanothermobacter thermoflexus ) Methanobacterium thermoflexum • Methanothermobacter soleniformans (Methanobacterium soleniformans)

[0160] • Methanofollis organophilus (Methanobrevibacter organophilus) Methanothermobacter wolfei ) Methanobacterium wolfei • Methanoreflexus songmohii

[0161] • Methanohalophilus Methanothrix sochngenii )

[0162] Optionally, the target cells are not pathogenic to the subject, for example when the method is a non-medical method. In one example, the method is a cosmetic method.

[0163] For example, the target cells are methanogenic cells, and optionally the subject is a livestock animal, preferably a ruminant, or a cow (e.g. a beef or dairy cow). By reducing methanogenic cells in such animals, in one embodiment, the present application can enhance the weight of the animal (e.g. enhance the meat yield from the animal) and / or enhance the yield of milk or another product such as fur or fat from the animal.

[0164] In one example, the target cell is selected from an Escherichia coli, Salmonella, and Campylobacter cell. In one example, the target cell is an Escherichia coli, Salmonella, or Campylobacter cell. In one example, each animal is a chicken (e.g., a broiler or a layer), and the target cell is a Salmonella or Campylobacter cell. In one example, each animal is a bovine (e.g., a beef or dairy cow), and the target cell is a methanogen cell.

[0165] In one example, the target cell is selected from a Mycoplasma (e.g., Mycoplasma Mycoplasma mycoides ) (e.g., Mycoplasma mycoides Mycoplasma mycoides subsp. Mycoides ), Mycoplasma leachii or Mycoplasma bovis Mycoplasma bovis ), Brucella abortus Brucella abortus ), Listeria monocytogenes Listeria monocytogenes ), Clostridium (e.g., Clostridium chauvoei Clostridium chauvoei ) or Clostridium septicum Clostridium septicum ), Leptospira (e.g., Leptospira interrogans (L. Leptospira ), Leptospira canicola ), Leptospira L. icterohaemorrhagiae ), Leptospira L. grippotyphosa ), Leptospira hardjo L. hardjo ), or Leptospira pomona L. Pomona ), Mannheimia haemolytica Mannheimia haemolytica ), Arcanobacterium pyogenes Trueperella pyogenes ), Mycobacterium bovis Mycobacterium bovis ), Campylobacter species (e.g., Campylobacter jejuni Campylobacter jejuni ) or Campylobacter coli Campylobacter coli ), Bacillus anthracis Bacillus anthracis ), Escherichia coli (e.g., Escherichia coli 0157:H7), or Pasteurella multocida Pasteurella multocida ) (e.g., Pasteurella multocida B:2, E:2, A:1, or A:3). In this example, optionally, the subject or animal is a livestock animal, e.g., a bovine, ovine, caprine, or chicken (preferably a bovine).

[0166] Optionally, for example, where the subject is an animal (e.g., a livestock animal or a wild animal), the target cells are animal-derived bacterial cells, e.g., cells of a species selected from the group consisting of Bacillus anthracis, Mycobacterium bovis (e.g., where the animal is a cow), Campylobacter spp. (e.g., where the animal is a poultry animal), Mycobacterium marinum (e.g., where the animal is a fish), Shiga-toxin producing E. coli (e.g., where the animal is a ruminant), Listeria spp. (e.g., where the animal is a cow or a sheep), Chlamydia abortus (e.g., where the animal is a sheep), Coxiella burnetii (e.g., where the animal is a cow, sheep, or goat), Salmonella spp. (e.g., where the animal is a poultry animal), Streptococcus suis (e.g., where the animal is a pig), and Corynebacterium (e.g., C. ulcerans) (e.g., where the animal is a cow).

[0167] In one example, a plurality of vector cells as herein (e.g., a vector cell of any configuration, aspect, example, or embodiment described herein) is administered to a subject, wherein the vector cells comprise DNA encoding an agent.

[0168] In one example, each animal is a chicken (e.g., a broiler or a layer), and the target cells are Salmonella or Campylobacter cells. In one example, each animal is a cow (e.g., a beef or dairy cow), and the target cells are methanogen cells.

[0169] Thus, in one embodiment, there is provided:

[0170] A method (e.g., non-medical or medical) for enhancing growth or weight of a subject, wherein the method comprises administering to the subject a plurality of vector cells, wherein the subject comprises bacterial target cells, and each vector cell is a bacterial cell comprising first episomal DNA encoding an antibacterial agent, the antibacterial agent being toxic to the target cells but not to the vector cells, the vector cells being capable of conjugative DNA transfer into the target cells for expression of the agent therein, wherein the first DNA is transferred from the vector cells into the target cells for expression therein to produce the antibacterial agent, thereby killing or reducing growth or proliferation of the target cells in the subject, and enhancing growth or weight of the subject.

[0171] Optionally, the target cell is a Salmonella cell. In one example, the target cell comprises a Salmonella enterica and / or a Salmonella typhimurium cell; optionally wherein the Salmonella enterica is Salmonella enterica subsp. enterica. Optionally, the method kills multiple different Salmonella enterica subsp. enterica serovars; optionally wherein each serovar is selected from the group consisting of typhimurium, enteritidis, wilsworth, montevideo, heidelberg, hadar, bongor, brenderup, infantis, kentucky, senftenberg, mbandaka, duck, agona, and dublin. Optionally, the method kills Salmonella enterica subsp. enterica serovars typhimurium, infantis, and enteritidis. Optionally, the method kills Salmonella enterica subsp. enterica serovars typhimurium and enteritidis. Optionally, the method kills Salmonella enterica subsp. enterica serovars typhimurium and infantis. Optionally, the method kills Salmonella enterica subsp. enterica serovars enteritidis and infantis. The most prevalent serovars in chickens are Salmonella enteritidis, Salmonella infantis, and Salmonella typhimurium. Generally, similar Salmonella serovars (Salmonella enteritidis and Salmonella typhimurium) are found in infected humans and chickens. By killing Salmonella in livestock animals, the present invention can be used to reduce the pool of animal-derived bacteria available for transmission to humans (e.g., through consumption of the livestock or products made therefrom, such as meat or dairy products for human consumption).

[0172] Advantageously, the vector cell is an Enterobacteriaceae cell, optionally an Escherichia coli cell. As evidenced by the examples below, the present inventors have surprisingly found that (i) the antibacterial agent can be effectively transferred from such cells to Salmonella target cells both in vivo and in vitro; and (ii) 100% killing of Salmonella is possible when the agent is transferred from the Enterobacteriaceae cell to the Salmonella cell by conjugation. Moreover, it was surprising that DNA encoding the guide nuclease antibacterial agent was able to kill 18 Salmonella species serovars, including clinically and zoonotically important typhimurium, infantis, and enteritidis. Optionally, the method kills Salmonella enterica subsp. enterica serovars typhimurium and enteritidis. Optionally, the Enterobacteriaceae cell is a cell of an Enterobacteriaceae species shown in Table 5.

[0173] Importantly, a reduction in target cells is observed in the GI tract of the subject. Thus, optionally, the method reduces target cells in the gastrointestinal tract of the animal; optionally, the method reduces target cells in the jejunum, ileum, colon, liver, spleen or caecum of the animal; optionally wherein the animal is a bird and the method reduces target cells in the caecum of the bird. This can be important for reducing the spread of animal-derived or otherwise harmful target strains of bacteria in the faeces of the subject, such as a livestock animal. Thus, in one example, the method is performed on a group of subjects (e.g. a herd or a flock, such as a herd of pigs or a flock of birds), wherein the spread of target species cells is reduced in the group. The inventors have also demonstrated this in a flock of poultry in which the nucleases are used to cleave multiple protospacer sequences in target cells, thereby killing the cells and reducing their spread in the flock, in which the nucleases are used to cleave multiple protospacer sequences in target cells, thereby killing the cells and reducing their spread in the flock.

[0174] Thus, in one example, the method is performed on a group (optionally a flock or a herd) of animals, wherein some or all of the animals comprise target cells, wherein the spread of the target species cells is reduced in the group; or wherein the spread from the group of animals to a second group of animals is reduced.

[0175] Optionally, the first DNA is comprised by a plasmid, such as wherein the plasmid comprises an RP4 transfer origin (oriT). The plasmid can be any type of plasmid disclosed herein.

[0176] The agent can be any antibacterial agent disclosed herein, preferably a guided nuclease programmed to cleave one or more target sequences in a target cell. Suitable nucleases can be TALENs, meganucleases, zinc finger nucleases, or Cas nucleases. For example, the agent comprises one or more components of a CRISPR / Cas system (e.g. a Cas nuclease and / or a guide RNA or crRNA) operable in a target cell to cleave a protospacer sequence comprised by the target cell, optionally wherein the target cell comprises a first strain and a second strain of a bacterial species, and each strain comprises a protospacer sequence, wherein cells of the strain are killed. For example, the system is operable to cleave at least 3 different protospacer sequences comprised by the genome of the cell. Optionally, each or some of the protospacer sequences are comprised by a pathogenicity island, which is comprised by the cell. As shown in the examples, this is highly effective for target cell killing. Optionally, the agent is operable to cleave a plurality of different protospacer sequences comprised by the genome of the target cell. Optionally, the agent comprises one or more components of a CRISPR / Cas system operable in a target cell to cleave at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different protospacer sequences comprised by the genome of the target cell (e.g. comprised by the chromosome of the target cell).

[0177] In one embodiment, the agent

[0178] (a) comprises a guided nuclease capable of recognizing and modifying a target nucleic acid sequence, wherein the target sequence is comprised by an endogenous chromosome or episome of a target cell, but not by a vector cell, wherein the nuclease modifies the chromosome or episome to kill the target cell or inhibit growth or proliferation of the target cell; and / or

[0179] (b) encodes a guide RNA or crRNA of a CRISPR / Cas system operable with a Cas nuclease in a target cell to cleave a protospacer sequence comprised by the target cell.

[0180] Also provided are:

[0181] A vector cell (optionally for use in a method of the application), wherein the cell is a bacterial cell comprising a first episomal DNA encoding an antibacterial agent, the antibacterial agent being toxic to a bacterial target cell but not to the vector cell, the vector cell being capable of conjugative transfer of DNA into the target cell for expression of the antibacterial agent therein, thereby killing the target cell, wherein the target cell is a Salmonella cell and the vector cell is an Enterobacteriaceae cell.

[0182] Also provided are:

[0183] A composition comprising a plurality of carrier cells for use in a method comprising administering the cells to a subject to treat an infection of pathogenic bacterial target cells, wherein each carrier cell is a bacterial cell comprising a first episomal DNA encoding an antibacterial agent that is toxic to the target cells but not to the carrier cells, the carrier cells being capable of DNA conjugative transfer into the target cells for expression of the agent therein, wherein the first DNA is transferred from the carrier cells into the target cells for expression therein to produce the antibacterial agent, thereby killing or reducing growth or proliferation of the target cells in the subject, wherein the target cells are Salmonella cells and the carrier cells are Enterobacteriaceae cells. In an alternative, the method treats or reduces symptoms of an infection of pathogenic target cells.

[0184] Any administration of cells to a subject herein can be by oral administration. Any administration of cells to a subject herein can preferably be by administration to the GI tract. Any administration of cells to a subject herein can be by systemic administration, intranasal administration, or inhalation administration.

[0185] Also provided are:

[0186] A non-medical method of killing animal-derived bacterial target cells in an animal, the method comprising administering to the animal a plurality of carrier cells, wherein each carrier cell is a bacterial cell comprising a first episomal DNA encoding an antibacterial agent that is toxic to the target cells but not to the carrier cells, the carrier cells being capable of DNA conjugative transfer into the target cells for expression of the agent therein, wherein the first DNA is transferred from the carrier cells into the target cells for expression therein to produce the antibacterial agent, thereby killing or reducing growth or proliferation of the target cells in the subject, wherein the target cells are Salmonella cells and optionally the carrier cells are Enterobacteriaceae cells.

[0187] The animal can be any animal disclosed herein, for example a livestock animal, a domesticated animal, or a wild animal (e.g., a bat or a bird).

[0188] Optionally, any method herein reduces Salmonella in the gastrointestinal tract of the subject. Optionally, the target cells comprise a different Salmonella species type that is killed.

[0189] In any aspect, configuration, example, concept, or embodiment, the carrier cell, target cell, or DNA is as defined in any other aspect, configuration, example, concept, or embodiment, respectively.

[0190] Also provided are:

[0191] DNA, wherein the DNA is capable of being introduced into a target cell, wherein the DNA encodes a plurality of guide RNAs or crRNAs of a CRISPR / Cas system, wherein the guide RNAs or crRNAs are operable with a Cas nuclease in the target cell to recognize a plurality of protospacer sequences comprised by a genome of the target cell, optionally wherein the target cell is a Salmonella cell or a cell of a species disclosed in Table 5; and

[0192] (a) the protospacer sequence comprises one or more pathogenicity island nucleotide sequences of a genome of a target cell;

[0193] (b) the protospacer sequence comprises one or more invasion gene sequences of a genome of a target cell;

[0194] (c) the protospacer sequence comprises one or more secretion system gene sequences of a genome of a target cell; and / or

[0195] (d) the protospacer sequence comprises one or more nucleotide sequences selected from the following genes of Salmonella (e.g., Salmonella enterica), or orthologs or homologs of the genes: avrA, sptP, sicP, sipA, sipD, sipC, sipB, sicA, invB, ssaE, sseA, sseB, sscA, sseC, sseD, sseE, sscB, sseF, sseG, mgtC, cigR, pipA, pipB, pipC, sopB and pipD (optionally selected from invB, sicP, sseE, pipA, pipB, pipC, hilA, marT and sopB ).

[0196] Ortholog: An ortholog is a gene, nucleotide, or protein sequence that is related to a second gene, nucleotide, or protein sequence by inheritance from a common ancestral DNA or protein sequence. The term ortholog can apply to the relationship between genes separated by an event or the relationship between genes separated by a genetic replication event.

[0197] Ortholog: An ortholog is a gene, nucleotide, or protein sequence that is related to a second gene, nucleotide, or protein sequence by inheritance from a common ancestral DNA or protein sequence. The term ortholog can apply to the relationship between genes separated by an event or the relationship between genes separated by a genetic replication event.

[0198] “Orthologs or homologs of the genes” means that the protospacer can be a sequence comprised by a gene of a target cell genome, wherein the gene is an ortholog or homolog of a Salmonella gene selected from the following: avrA, sptP, sicP, sipA, sipD, sipC, sipB, sicA, invB, ssaE, sseA, sseB, sscA, sseC, sseD, sseE, sscB, sseF, sseG, mgtC, cigR, pipA, pipB, pipC, sopB and pipD (optionally selected from invB, sicP, sseE, pipA, pipB, pipC, hilA, marT and sopB ).

[0199] Optionally, the DNA is a first DNA used in the method of the application. Preferably, the target cell is a Salmonella cell, for example a Salmonella enterica cell, for example a Salmonella enterica enterica subsp. enterica serovar cells.

[0200] Optionally, any Salmonella herein is Salmonella enterica enterica subsp. enterica serovar typhimurium strain LT2.

[0201] Optionally, the target cell is a cell of a species other than Salmonella species disclosed in Table 5, and the sequence of (d) is a gene sequence of an ortholog or homolog selected from the following of Salmonella (e.g., Salmonella enterica): avrA, sptP, sicP, sipA, sipD, sipC, sipB, sicA, invB, ssaE, sseA, sseB, sscA, sseC, sseD, sseE, sscB, sseF, sseG, mgtC, cigR, pipA, pipB, pipC, sopB and pipD (optionally selected from Exemplary plasmids incP and incP ).

[0202] The DNA can comprise one or more CRISPR spacers, wherein each spacer consists of a nucleotide sequence of a secretion system gene (optionally a type III protein secretion system or a secretion system of SPI-1 or SPI-2 of Salmonella), or a T3SS locus gene, or differs from it by at most 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides.

[0203] Optionally, the DNA (e.g., of the vector cell) encodes a plurality of guide RNAs or crRNAs of a CRISPR / Cas system, wherein the guide RNAs or crRNAs are operable with a Cas nuclease in a target cell to recognize a plurality of protospacer sequences comprised by a genome of the target cell, wherein the target cell is a Salmonella cell, and the protospacer sequences comprise one or more nucleotide sequences of a gene selected from incP and IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, Inc10, Inc11, For example, the protospacer sequences comprise nucleotide sequences of genes IncA, IncB, IncC, IncH, IncIa, IncIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, and IncS, IncT In one example, the DNA further encodes a Cas, for example Cas9, Cas3, Cpfl, Casl2, Casl3, CasX, or CasY. In one embodiment, the Cas is a Type I, II, III, IV, V, or VI Cas, preferably a Type I or II Cas. In one example, the DNA further encodes Cas3 and homologous cascade proteins (e.g., CasA, B, C, D, and E). Optionally, the Cas (and cascade present) is an E. coli Cas (and cascade).

[0204] Optionally, the gene of the target cell encodes a chaperone protein or a secreted effector protein, for example such a protein encoded by a pathogenicity island or a type III protein secretion system (optionally a T3SS locus, for example the secretion systems of SPI-1 and SPI-2 of Salmonella).

[0205] The DNA can comprise one or more CRISPR spacer regions, wherein each spacer region consists of 20-40, 25-35 or 30-35 contiguous nucleotides of a gene comprised by the genome of the target cell; for example,

[0206] (a) a gene selected from the group consisting of: IncW IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, Inc10, Inc11, and IncA, IncB, IncC, IncH, IncIa, IncIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, a homologue or orthologue thereof;

[0207] (b) a gene comprised by a pathogenicity island, the pathogenicity island being comprised by the genome of the target cell;

[0208] (c) a secretion system (for example a type III protein secretion system) gene comprised by the genome of the target cell.

[0209] Optionally, the DNA is comprised by a plasmid comprising a transfer origin (oriT) and a replication origin (oriV), the replication origin being operable for DNA replication in a bacterial host cell. Optionally, the first DNA is comprised by a plasmid, wherein the plasmid comprises an RP4 transfer origin (oriT) and / or a p15A replication origin.

[0210] Optionally, the DNA comprises SEQ ID NO: 15, optionally wherein the DNA is comprised by a plasmid in a carrier bacterial cell for conjugation with a Salmonella target cell. In one example, the DNA of the application is comprised by a conjugative plasmid or a phagemid.

[0211] In one example, the DNA comprises a CRISPR repeat sequence and a spacer sequence, wherein

[0212] (a) the repeat sequences each comprise SEQ ID NO: 16; and / or

[0213] (b) the spacer sequence comprises 1, 2 or 3 sequences selected from the group consisting of SEQ ID NOs: 17-19 and the complement sequences thereof;

[0214] Optionally wherein the DNA is comprised by a plasmid in a carrier bacterial cell for conjugation with a Salmonella target cell.

[0215] Optionally, the DNA comprises (optionally in 5' to 3' order) SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. Optionally, the DNA comprises one or more (e.g., at least 3) spacer sequences set forth in Table 6.

[0216] In one example, the DNA encodes Cas3 and optionally one or more cascade proteins (e.g., one or more of CasA, B, C, D, and E). In one embodiment, the first DNA encodes Cas3 and CasA, B, C, D, and E. In one embodiment, the first DNA encodes E. coli Cas3 and CasA, B, C, D, and E. Optionally, the guide nuclease (e.g., Cas3) is a Type I-A, -B, -C, -D, -E, -F, or -U Cas.

[0217] In one example, the agent in any configuration, example, option, or embodiment herein comprises one or more components of a CRISPR / Cas system operable in a target cell to cleave a protospacer sequence comprised by the target cell.

[0218] In one example, the system is operable to cleave at least 3 different protospacer sequences comprised by the genome of a target cell. In one embodiment, each or some of the protospacer sequences are comprised by a pathogenicity island, the pathogenicity island being comprised by the target cell.

[0219] In one example, the agent in any configuration, example, option, or embodiment herein

[0220] (a) a guide nuclease comprising capable of recognizing and modifying a target nucleic acid sequence, wherein the target sequence is comprised by an endogenous chromosome or episome of a target cell, but not by a vector cell, wherein the nuclease modifies the chromosome or episome to kill or inhibit growth or proliferation of the target cell; and / or

[0221] (b) a guide RNA or crRNA encoding a CRISPR / Cas system operable with a Cas nuclease in a target cell to cleave a protospacer sequence comprised by the target cell.

[0222] Optionally, the DNA comprises a constitutive promoter for expression of the guide RNA or crRNA. Optionally, the DNA comprises a constitutive promoter for expression of a Cas nuclease operable with the guide RNA or crRNA in a target cell to modify (e.g., cleave) a protospacer sequence of the genome of the target cell.

[0223] Optionally, the Cas, Cascade protein, gRNA, and crRNA are E. coli K12 (MG1655) Cas, Cascade protein, gRNA, and crRNA, respectively. Optionally, the DNA lacks nucleotide sequences encoding Casl and Cas2 proteins.

[0224] The present application also relates to vector bacteria encoding a desired protein or RNA (e.g., encoding an antimicrobial agent) and methods of use. In embodiments, an agent can be transferred into a target cell by conjugation between a vector cell (to which the agent is non-toxic) and the target cell, whereby the agent is toxic to the target cell and kills the target cell. In other embodiments, the growth or proliferation of the target cell is reduced (e.g., by at least 40, 50, 60, 70, 80, or 90% as compared to growth in the absence of the agent). Each vector cell comprises episomal DNA encoding an antibacterial agent that is toxic to a target bacterial cell, but is non-toxic (or less toxic) to the vector cell. The present application can be applied, for example, to control or kill target bacteria that are pathogenic to humans, animals, or plants. The present application can be applied, for example, to control or kill animal-derived target bacteria contained by an animal (e.g., a livestock animal). For example, the vector cell can comprise a drug for treating or preventing a disease or condition in a human or animal; a growth promoter for administration to an animal to promote its growth; an animal-derived bacteria-killing agent in an animal; a pesticide for administration to livestock; a pesticide to be applied to a plant; or a plant fertilizer.

[0225] One advantage of the present application is that the vector cell can be used as a production cell, in which DNA encoding an antibacterial agent can be replicated. Another advantage of one example of the present application is that further replication of the DNA can be avoided in a target cell that does not comprise one or more factors required for such replication. Thus, one can envision a scenario in which the factors are present in the vector cell (and DNA encoding the agent is replicated to provide many copies for subsequent conjugative transfer into a target cell), and in the target cell, the DNA is not further replicated, which can be useful to contain the effects of the antibacterial agent, for example, in the environment or in the body of a human or animal or in a plant (or on a plant). Containment can be desirable to avoid unwanted killing of non-target cells, or to provide general control over administration and / or killing activity scenarios.

[0226] The first DNA can replicate in the carrier cell (but not in the target cell), in which case it is important that the agent is not toxic to the carrier cell, while it is toxic to the target cell. In certain embodiments, the present application uses sequence-specific killing of the target cell to achieve this selectivity. To this end, in one example, the first DNA encodes a guide nuclease that can operate in the target cell to recognize and cleave a target sequence of the target cell's chromosome, thereby killing the cell or reducing its growth or proliferation. This advantageously exceeds the use of other types of toxic agents that are less discriminating in their action, capable of killing several species or strains (e.g., potentially toxic to the carrier cell to some extent as well). By using a guide nuclease (e.g., a TALEN or a Cas nuclease), these can be programmed to recognize a target sequence that is present in the genome of the target cell (e.g., comprised by a chromosome or episome of the target cell), but not in the genome of the carrier cell.

[0227] Thus, in this case, replication of the first DNA can occur freely in the carrier cell, without the risk of killing the cell or reducing its growth or proliferation due to replication of the encoded agent and the sequence encoding the agent. Thus, in one example where the first DNA encodes a guide nuclease, the guide nuclease is capable of recognizing and cleaving a target nucleic acid sequence comprised by the genome (e.g., chromosome) of the target cell, wherein the target cell is not present in the carrier cell.

[0228] One particularly useful example is where the first DNA encodes a Cas nuclease (e.g., Cas9 or Cas3) that can operate with a guide RNA or crRNA in the target cell, wherein the RNA is operable to guide the Cas to a target sequence, wherein the Cas modifies (e.g., cleaves) the target sequence and kills the target cell or inhibits the target cell growth or proliferation. In one embodiment, the first DNA encodes the Cas and the guide RNA or crRNA. In another embodiment, the first DNA encodes the guide RNA or crRNA, but not the cognate Cas. In this embodiment, the RNA is operable in the target cell with an endogenous Cas encoded by the genome of the target cell, wherein the RNA is operable to guide the Cas to a target sequence, wherein the Cas modifies (e.g., cleaves) the target sequence and kills the target cell or inhibits the target cell growth or proliferation. In this sense, the agent can comprise components of a CRISPR / Cas system (e.g., Cas nuclease, Cascade Cas, crRNA, guide RNA or tracrRNA). Thus, the present application effectively recognizes the benefit of using an antibacterial agent that acts through target recognition in the target cell but not the carrier cell, which opens the ability for the first DNA to replicate freely in the carrier cell without significant toxicity to the carrier cell.

[0229] IncS, IncT

[0230] Any method of delivery of an agent, such as a CRISPR-Cas system (or components thereof) can be by bacterial conjugation - the natural process by which donor bacteria transfer DNA from themselves to recipient bacteria. Donor bacteria are carefully engineered with surface structures through which they deliver DNA - which can be thought of like a syringe or straw. The donor pilus binds to the surface of the recipient and this event triggers the process of DNA transfer. Plasmids are amenable to this conjugation process, wherein the plasmid comprises DNA encoding an agent of the application.

[0231] DNA transfer by conjugation can only occur for'susceptible recipients', but generally not for recipients carrying a similar type of plasmid. Because conjugation is via a pilus bridge, it is possible that the bridge does not attach itself to the recipient, but to the donor bacterium. This can lead to an ineffective cycle of plasmid DNA transfer to itself. Therefore, plasmids naturally encode an incompatibility factor. One is a surface arrayed protein which prevents binding of the pilus to bacteria displaying this surface protein, such as itself or any other bacteria carrying the same plasmid. Additionally, plasmids naturally encode another incompatibility system which tightly regulates the number of plasmid copies within the bacterium. Therefore, if a conjugation event manages to escape the surface repulsion and starts to transfer DNA by conjugation, the recipient will prevent establishment of the plasmid because it already maintains the current copy number and does not accept and maintain further unwanted additional copies.

[0232] In one example of the application, the DNA encoding the agent is comprised by a plasmid. In one embodiment, the plasmid is a member of a plasmid incompatibility group, wherein the target cell does not comprise a plasmid of the group. Optionally, the plasmid of the application is a member of incompatibility group P (i.e. the plasmid is a P plasmid). Salmonella rarely carry IncW plasmids, so when the target cell is a Salmonella cell, such a incP plasmid is useful. For example, within the Enterobacteriaceae, the following is a non-exclusive list of potential plasmids which can be used for delivery: incF1 IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, Inc10, Inc11, IncA, IncB, IncC, IncH, IncIa, IncIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT and / or IncW Therefore, optionally, the target cell is an Enterobacteriaceae cell and the DNA of the application is comprised by a plasmid, wherein the plasmid is selected from IncFI, ​ ​ and ​ plasmids.

[0233] ​In one example, the vector cell of the present invention comprises two or more plasmids, each plasmid containing the DNA of the present invention encoding an antimicrobial agent, wherein a first plasmid is a member of a first incompatibility group, wherein the target cell does not contain plasmids of the first group, and wherein a second plasmid is a member of a second incompatibility group, wherein the target cell does not contain plasmids of the second group. For example, the vector cell may contain a first crRNA or guide RNA encoding an anti-target cell CRISPR-Cas system or a component thereof (e.g., a first crRNA or guide RNA encoding a first prespacer region sequence targeting the target cell genome). ​ The plasmid, wherein the vector cell further comprises a plasmid encoding an anti-target cell CRISPR-Cas system or a component thereof (e.g., a second crRNA or guide RNA encoding a second prespacer sequence targeting the target cell genome). ​ The plasmid contains different nucleotide sequences in the anterior spacer region. For example, the anterior spacer region is contained by different genes in the target cell genome. For example, the anterior spacer region is contained by one or more pathogenic islands in the target cell genome. Optionally, the target cell is an Enterobacteriaceae cell. Optionally, the vector cell contains a set of plasmids comprising 2, 3, 4, 5, 6 or more plasmids of different types, wherein each plasmid is capable of binding and transfecting into the target cell, wherein the plasmids encode different components of different reagents or antibacterial agents. For example, the plasmids encode different cRNAs or gRNAs that target different anterior spacer regions contained in the target cell genome. For example, the set of plasmids contains a maximum of n There are several different types of plasmids, of which plasmids are the most numerous. n Members of different incompatible groups, for example, selected from ​ ​ ​ and ​ Groups. For example, n = 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0234] For example, the vector cell comprises (i) a first plasmid encoding a first type of CRISPR / Cas system or encoding components of the system, the first type of CRISPR / Cas system targeting a first protospacer included by the target cell genome; and (ii) a second plasmid encoding a second type of CRISPR / Cas system or encoding components of the system, the second type of CRISPR / Cas system targeting a second protospacer included by the target cell genome, wherein the first type and the second type are different. For example, the first type is a type I system, and the second type is a type II system (e.g., the first plasmid encodes Cas3, Cascade, and a crRNA or guide RNA, which can operate with Cas3 and Cascade in the target cell to modify the first protospacer; and the second plasmid encodes Cas9 and a crRNA or guide RNA, which can operate with Cas9 in the target cell to modify the second protospacer). In an alternative, Cas3 and Cascade are encoded by endogenous target cell genes, wherein the first plasmid encodes a crRNA or guide RNA, which can operate with endogenous Cas3 and Cascade in the target cell to modify the first protospacer. In an alternative, Cas9 is encoded by an endogenous target cell gene, wherein the second plasmid encodes a crRNA or guide RNA, which can operate with endogenous Cas9 in the target cell to modify the second protospacer. Optionally, Cas3 and Cascade are encoded by endogenous genes of the target cell, and Cas9 is encoded by the second plasmid.

[0235] Instead of a type I and a type II system, in an embodiment, the application alternatively provides a first plasmid encoding a type I CRISPR / Cas system (or components thereof, e.g., Cas3 or a crRNA or gRNA), and a second plasmid encoding a type III CRISPR / Cas system (or components thereof). Instead of a type I and a type II system, in an embodiment, the application alternatively provides a first plasmid encoding a type I CRISPR / Cas system (or components thereof), and a second plasmid encoding a type IV CRISPR / Cas system (or components thereof). Instead of a type I and a type II system, in an embodiment, the application alternatively provides a first plasmid encoding a type I CRISPR / Cas system (or components thereof), and a second plasmid encoding a type V CRISPR / Cas system (or components thereof). Instead of a type I and a type II system, in an embodiment, the application alternatively provides a first plasmid encoding a type I CRISPR / Cas system (or components thereof), and a second plasmid encoding a type VI CRISPR / Cas system (or components thereof).

[0236] Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type II CRISPR / Cas system (or components thereof, e.g., Cas9 or crRNA or gRNA), and a second plasmid encoding a Type III CRISPR / Cas system (or components thereof). Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type II CRISPR / Cas system (or components thereof), and a second plasmid encoding a Type IV CRISPR / Cas system (or components thereof). Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type II CRISPR / Cas system (or components thereof), and a second plasmid encoding a Type V CRISPR / Cas system (or components thereof). Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type II CRISPR / Cas system (or components thereof), and a second plasmid encoding a Type VI CRISPR / Cas system (or components thereof).

[0237] Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type V CRISPR / Cas system (or components thereof, e.g., Casl2a or crRNA), and a second plasmid encoding a Type III CRISPR / Cas system (or components thereof). Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type V CRISPR / Cas system (or components thereof), and a second plasmid encoding a Type IV CRISPR / Cas system (or components thereof). Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type V CRISPR / Cas system (or components thereof), and a second plasmid encoding a Type V CRISPR / Cas system (or components thereof). Instead of a Type I and Type II system, in an embodiment, the present application alternatively provides a first plasmid encoding a Type V CRISPR / Cas system (or components thereof), and a second plasmid encoding a Type VI CRISPR / Cas system (or components thereof).

[0238] Instead of Type I and Type II systems, in one embodiment, the application alternatively provides a first plasmid and a second plasmid each encoding a Type I CRISPR / Cas system (or components thereof). Instead of Type I and Type II systems, in one embodiment, the application alternatively provides a first plasmid and a second plasmid each encoding a Type II CRISPR / Cas system (or components thereof). Instead of Type I and Type II systems, in one embodiment, the application alternatively provides a first plasmid and a second plasmid each encoding a Type III CRISPR / Cas system (or components thereof). Instead of Type I and Type II systems, in one embodiment, the application alternatively provides a first plasmid and a second plasmid each encoding a Type IV CRISPR / Cas system (or components thereof). Instead of Type I and Type II systems, in one embodiment, the application alternatively provides a first plasmid and a second plasmid each encoding a Type V CRISPR / Cas system (or components thereof). Instead of Type I and Type II systems, in one embodiment, the application alternatively provides a first plasmid and a second plasmid each encoding a Type VI CRISPR / Cas system (or components thereof).

[0239] Optionally, the plasmids are members of different incompatible groups, e.g., selected from the group of ​ IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Incl, IncA, IncB, IncC, IncH, IncIa, InclIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT and IncW In one example here, the target cell is an Enterobacteriaceae cell.

[0240] Embodiments

[0241] Thus, as an example, the application provides the following embodiments.

[0242] 1. A carrier bacterial cell comprising a first episomal DNA, said DNA comprising a nucleic acid of interest (NSI) encoding a protein of interest (POI) or a RNA of interest (ROI) for expression of the POI or ROI in a target bacterial cell, said carrier cell being capable of conjugal transfer of the DNA into the target cell for expression of the POI, ROI or agent therein, wherein

[0243] (a) the carrier cell comprises a second DNA different from the first DNA, wherein the second DNA comprises or encodes a first factor required for replication of the first DNA;

[0244] (b) the first DNA does not comprise or encode the first factor, wherein the first DNA is non-self-replicating in the absence of the first factor, but is capable of replication in the carrier cell in the presence of the first factor provided by the second DNA;

[0245] (c) wherein the carrier cell comprises a gene encoding one or more conjugation factors sufficient to perform a first DNA conjugative transfer into a target bacterial cell.

[0246] In a first alternative, embodiment 1 provides:-

[0247] A carrier bacterial cell comprising first episomal DNA, the DNA comprising a nucleic acid of interest (NSI) encoding a protein of interest (POI) or a RNA of interest (ROI) for expression of the POI or ROI in a target bacterial cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression of the POI or ROI therein.

[0248] In a second alternative, embodiment 1 provides:-

[0249] A carrier bacterial cell comprising first episomal DNA, the DNA encoding an antibacterial agent toxic to the target bacterial cell but not to the carrier cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression of the agent therein.

[0250] In one example, the agent comprises a guided nuclease capable of recognising and cleaving a target nucleic acid sequence comprised by the target cell genome, wherein the target sequence is not comprised by the carrier cell. Thus, in this sense, the antibacterial agent is toxic to the target bacterial cell but not to the carrier cell.

[0251] For example, there is provided:-

[0252] A carrier bacterial cell comprising first episomal DNA, the DNA encoding an antibacterial agent toxic to the target bacterial cell but not to the carrier cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression of the agent therein, wherein the agent is a component of a CRISPR / Cas system operable in the target cell to modify a target nucleic acid sequence comprised by the target cell genome (e.g. by a target cell chromosome).

[0253] Another example provides:-

[0254] A carrier bacterial cell comprising first episomal DNA, the DNA encoding an antibacterial agent toxic to the target bacterial cell but not to the carrier cell, the carrier cell being capable of conjugative transfer of the DNA into the target cell for expression of the agent therein, wherein

[0255] (a) the carrier cell comprises second DNA distinct from the first DNA, wherein the second DNA comprises or encodes a first factor required for replication of the first DNA;

[0256] (b) the first DNA does not comprise or encode the first factor, wherein the first DNA is non-self-replicating in the absence of the first factor, but is capable of replication in the vector cell in the presence of the first factor provided by the second DNA;

[0257] (c) wherein the vector cell comprises a gene encoding one or more conjugation factors sufficient to perform a conjugative transfer of the first DNA into a target bacterial cell.

[0258] One example also provides:

[0259] A vector bacterial cell comprising a plasmid comprising first DNA encoding a guide nuclease capable of recognizing and modifying a target nucleic acid sequence, the vector cell being capable of conjugative transfer of the plasmid into a target cell for expression of the nuclease therein, wherein the target sequence is comprised by an endogenous chromosome or episome of the target cell but not by the vector cell, wherein the nuclease is capable of modifying (e.g. cleaving) the chromosome or episome to kill or inhibit growth or proliferation of the target cell.

[0260] Optionally, the nuclease is a Cas nuclease, a meganuclease, a zinc finger nuclease or a TALEN. Optionally, the nuclease is a Cas nuclease of a Type I, II, III, IV or V CRISPR system.

[0261] One example also provides:

[0262] A vector bacterial cell comprising a plasmid comprising first DNA encoding a component of a CRISPR / Cas system capable of recognizing and modifying a target nucleic acid sequence, the vector cell being capable of conjugative transfer of the plasmid into a target cell for expression of the component therein, whereby the expressed component constitutes part of the system in the target cell, wherein the target sequence is comprised by an endogenous chromosome or episome of the target cell but not by the vector cell, wherein the system is capable of modifying (e.g. cleaving) the chromosome or episome to kill or inhibit growth or proliferation of the target cell.

[0263] In one aspect, the component is a guide RNA or crRNA capable of hybridizing to a target sequence of the target cell. The system can be a Type I, II, III, IV or V CRISPR system.

[0264] Optionally,

[0265] (a) the vector cell comprises second DNA different from the first DNA, wherein the second DNA comprises or encodes the first factor required for replication of the first DNA;

[0266] (b) the first DNA does not comprise or encode the first factor, wherein the first DNA is non-self-replicating in the absence of the first factor, but is capable of replication in the vector cell in the presence of the first factor provided by the second DNA;

[0267] (c) wherein the vector cell comprises a gene encoding one or more conjugation factors sufficient to perform a first DNA conjugative transfer into a target bacterial cell.

[0268] The second DNA can be comprised by a chromosome or an episome (e.g., a plasmid) of the vector cell.

[0269] Any of the methods of the application can use a vector cell of any of these examples.

[0270] Advantageously, the cell is used to treat or prevent infection of target cells in a human or animal subject (e.g. a chicken, a cow, a pig, a fish or a shellfish). Advantageously, the carrier cell is a cell of a species that is a probiotic to the subject or a probiotic to a human or animal (e.g. a chicken). For example, the carrier cell is a probiotic Escherichia coli cell. For example, the carrier cell is a probiotic Bacillus cell. In one example, the target cell is a cell of a species that is pathogenic to the subject or pathogenic to a human or animal (e.g. a chicken). Advantageously, the first DNA encodes one or more guide RNAs or one or more crRNAs that are capable of hybridising to a respective target nucleic acid sequence in the target cell, wherein the target sequence is comprised by an endogenous chromosome and / or endogenous episome of the target cell. For example, the first DNA encodes 2, 3, 4, 5, 6, 7, 7, 9 or 10 (or more than 10) different gRNAs or different crRNAs that hybridise to respective target sequences, wherein the target sequences are different from one another. For example, 3 different gRNAs or crRNAs are encoded by the first DNA. For example, 2 different gRNAs or crRNAs are encoded by the first DNA. For example, 3 different gRNAs or crRNAs are encoded by the first DNA. For example, 4 different gRNAs or crRNAs are encoded by the first DNA. For example, 3 different gRNAs or crRNAs are encoded by the first DNA. For example, 5 different gRNAs or crRNAs are encoded by the first DNA. For example, 6 different gRNAs or crRNAs are encoded by the first DNA. For example, 7 different gRNAs or crRNAs are encoded by the first DNA. For example, 8 different gRNAs or crRNAs are encoded by the first DNA. For example, 9 different gRNAs or crRNAs are encoded by the first DNA. For example, 10 different gRNAs or crRNAs are encoded by the first DNA. For example, 11 different gRNAs or crRNAs are encoded by the first DNA. For example, 12 different gRNAs or crRNAs are encoded by the first DNA. For example, 13 different gRNAs or crRNAs are encoded by the first DNA. In one example, the target cell is a Salmonella cell (e.g. wherein the subject is a chicken). In one example, the target cell is an Escherichia coli cell. In one example, the target cell is a Campylobacter cell (e.g. wherein the subject is a chicken). In one example, the target cell is an Edwardsiella Edwardsiella ) cell (e.g. wherein the subject is a fish or shellfish, e.g. a catfish or prawn or shrimp). In one example, the target cell is an Escherichia coli cell.

[0271] In one alternative herein, the carrier cell and the target cell are archaeal cells. In one alternative herein, the carrier cell and the target cell are yeast cells, and “conjugation” is instead understood to refer to yeast “mating”, and for example the second DNA is comprised by a chromosome of the carrier yeast cell.

[0272] In one preferred example, the NOI encodes an antibacterial agent that is toxic to the target bacterial cell, but not to the carrier cell. In one example, the POI is an antibiotic agent, an antibody, an antibody chain, or an antibody variable domain. In one example, the ROI is a guide RNA or crRNA that is operable with a cognate Cas (e.g., a Cas nuclease that targets and cleaves a protospacer sequence comprised by a chromosome or episome of the target cell) in the target cell. In one example, the RNA is an siRNA that is capable of hybridizing to an endogenous target nucleic acid sequence of the target cell to silence its transcription and / or translation.

[0273] The carrier cell comprises a chromosome or a second episomal DNA that is different from the first DNA. Thus, for example, the second DNA is comprised by a chromosome of the carrier cell. In another example, the second DNA is comprised by a plasmid of the carrier cell.

[0274] 2. The carrier cell of embodiment 1, wherein

[0275] (d) the first DNA lacks a component required for conjugal transfer of the first DNA into the target bacterial cell; and

[0276] (e) the carrier cell comprises the component, wherein the component is comprised or encoded by a second DNA or a third DNA, the second DNA or third DNA being comprised by the carrier cell.

[0277] The third DNA can be comprised by a chromosome or an episome (e.g., a plasmid). When explicitly recited in embodiment 2, the component refers to a component required for conjugal transfer, as explicitly recited by the context.

[0278] 3. The carrier cell of embodiment 2, wherein the component is comprised by an Mpf or Dtr module.

[0279] For example, the module is an RK2, RP4, or R6K tra module. Optionally, the component is comprised by an Mpf (mating pair formation) module, e.g., an RK2 or RP4 tra1 module or homolog thereof. Optionally, the component is comprised by a Dtr (DNA transfer replication) module, e.g., an RK2 or RP4 tra2 module or homolog thereof.

[0280] 4. The carrier cell of embodiment 2, wherein the component is encoded by an operon of an Mpf (e.g., tra1) or Dtr (e.g., tra2) module.

[0281] For example, the module is an RK2, RP4 or R6K tra module. Optionally, the module is an Mpf (mating pair formation) module, such as an RK2 or RP4 tra1 module or homologue thereof. Optionally, the module is a Dtr (DNA transfer replication) module, such as an RK2 or RP4 tra2 module or homologue thereof.

[0282] The Mpf operon of RP4 or RK2 is the tra2 operon (comprising the trb genes trbBCDEFGHIJKL) along with the gene traF. The traF gene is included in the tra1 operon in RK2 or RP4 (along with traJXIHG). Thus, in one embodiment, the component is a gene selected from the group consisting of trbB, trbC, trbD, trbE, trbF, trbG, trbH, trbI, trbJ, trbK, trbL and traF. In one embodiment, the first DNA lacks two or more genes selected from the group consisting of RP4 trbB, trbC, trbD, trbE, trbF, trbG, trbH, trbI, trbJ, trbK, trbL and traF or homologues thereof. In one embodiment, the first DNA lacks two or more genes selected from the group consisting of RK2 trbB, trbC, trbD, trbE, trbF, trbG, trbH, trbI, trbJ, trbK, trbL and traF or homologues thereof. In one example, the component is a traK, traL or traM gene. In one embodiment, the first DNA lacks two or more genes selected from the group consisting of RP4 traK, traL and traM or homologues thereof. In one embodiment, the first DNA lacks two or more genes selected from the group consisting of RK2 traK, traL and traM or homologues thereof. Optionally, in these embodiments, the first DNA is comprised by a RP4 or RK2 type plasmid.

[0283] The R6K Mpf is encoded by the sltX1tivB1B2B3-4B5B6B7B8B9B10B11 operon which also includes the clpX1 of the Dtr module. Thus, in one embodiment, the component is a gene comprised by the RK6 sltX1tivB1B2B3-4B5B6B7B8B9B10B11 operon. In one embodiment, the component is the clpX1 of the RK6 Dtr module. In one embodiment, the component is a gene selected from the group consisting of clpX1 and dtrX1rlxX1. In one embodiment, the component is a gene selected from the group consisting of RK6 clpX1 and dtrX1rlxX1 or homologues thereof. Optionally, in these embodiments, the first DNA is comprised by a RK6 type plasmid.

[0284] The vector cell of any preceding embodiment, wherein the vector cell chromosome and / or episome of the vector cell (other than the episome comprising the first DNA) comprises an expressible tra1 and / or tra2 module or homologue thereof.

[0285] Any episome herein can be a plasmid.

[0286] 5. The vector cell of any preceding embodiment, wherein the vector cell chromosome and / or episome of the vector cell (other than the episome comprising the first DNA) comprises an expressible operon of a tra1 and / or tra2 module or homologue thereof.

[0287] 6. The vector cell of any one of embodiments 3 to 6, wherein the component is a tra1 component.

[0288] 7. The vector cell of any one of embodiments 3 to 6, wherein the component is a tra2 component.

[0289] 8. The vector cell of any one of embodiments 2 to 7, wherein the vector cell chromosome encodes the first factor and comprises the component.

[0290] 9. The vector cell of any preceding embodiment, wherein the first factor is rep a protein, optionally wherein the protein is encoded by pir or trfA or a homologue thereof.

[0291] 10. The vector cell of any preceding embodiment, wherein the first DNA is comprised by an RK2 or R6K plasmid.

[0292] 11. The vector cell of any preceding embodiment, wherein the first DNA comprises oriV or a homologue thereof of an RK2 or R6K plasmid.

[0293] 12. The vector cell of any preceding embodiment, wherein the first DNA comprises oriT or a homologue thereof of an RK2 or R6K plasmid.

[0294] 13. The vector cell of any preceding embodiment, wherein the first DNA is comprised by a plasmid.

[0295] 14. The vector cell of any preceding embodiment, wherein the second DNA is comprised by a plasmid or by a chromosome of the vector cell.

[0296] 15. A vector cell of any of the foregoing embodiments, wherein the reagent comprises one or more components of a CRISPR / Cas system, the CRISPR / Cas system being operable in a target cell to cleave an anterior spacer sequence contained in the target cell, for example, wherein the anterior spacer sequence is contained in a cell chromosome.

[0297] In one embodiment, the cleavage kills the target cells. In another embodiment, the cleavage inhibits the growth or proliferation of the target cells.

[0298] 16. A vector cell of any of the foregoing embodiments, wherein the reagent encodes a guide RNA or crRNA of a CRISPR / Cas system that can be operated with a Cas nuclease in the target cell to cleave an anterior spacer sequence contained in the target cell, for example, wherein the anterior spacer sequence is contained in the cell chromosome.

[0299] In one instance, the target cells were Salmonella cells, and the anterior septum was composed of… pipA, pipB, pipC, hilA, sicP, mart or sopB The gene is contained within. In one instance, the anterior septum is contained within a gene, which is a Salmonella spp. sicP, sseF, pipA, pipB, pipC, hilA, sicP, mart or sopB Homologous or orthologous genes.

[0300] 17. A vector cell of any of the foregoing embodiments, wherein the first DNA contains a gene encoding a product, wherein the product is essential for the survival or proliferation of the vector cell when in an environment lacking the product, wherein the vector cell chromosome does not contain an expressible gene encoding the product, and optionally the first DNA is the only additional DNA encoding the product contained by the vector cell.

[0301] 18. The vector cell of embodiment 17, wherein the gene is selected from... aroA, argH, hisD, leuB, lysA, metB, proC, thrC, pheA, tyrA, trpC and pflA A gene; or wherein the gene is an antitoxin gene, and optionally the first DNA encodes a homologous toxin.

[0302] 19. The carrier cell of any of the foregoing embodiments, wherein the carrier cell is *Escherichia coli* (e.g., Nissle, F18, or S17 *Escherichia coli* strains), *Bacillus* (e.g., *Bacillus subtilis*), *Enterococcus* (… Enterococcus ) or Lactobacillus spp. Lactobacillus )cell.

[0303] Optionally, the carrier cell is a cell of a symbiotic bacterial strain (e.g., a symbiotic Escherichia coli strain) of humans, chickens, pigs, sheep, cattle, fish (e.g., catfish or salmon), or shellfish (e.g., shrimp or lobster).

[0304] 20. The vector cell of any preceding embodiment, wherein the vector cell is for administration to the microbiota of a human or animal subject for medical use.

[0305] For example, the medical use is for the treatment or prevention of a disease disclosed herein. For example, the medical use is for the treatment or prevention of a condition disclosed herein.

[0306] 21. The vector of embodiment 20, wherein the medical use is for the treatment or prevention of a disease or condition mediated by the target cell.

[0307] 22. The vector of any one of embodiments 1 to 20, for administration to an animal to enhance the growth or weight of the animal.

[0308] In an alternative, the administration is to a human for enhancing the growth or weight of the human.

[0309] Optionally, the enhancement is not a medical therapy. Optionally, the enhancement is a medical therapy.

[0310] 23. The vector of any one of embodiments 20 to 22, wherein the use comprises administering a plurality of the vector cells to the microbiota (e.g., gut microbiota) of a subject, wherein the microbiota comprises the target cell, and the first DNA is transferred into the target cell for expression therein to produce an antibacterial agent to kill or reduce the growth or proliferation of the target cell in the subject.

[0311] 24. DNA encoding an antibacterial agent that is toxic to a target bacterial cell but not to a vector cell, wherein the first DNA comprises an origin of replication but does not comprise or encode a first factor required for replication of the first DNA, and wherein the first DNA comprises an origin of transfer but lacks a component required for conjugal transfer of the first DNA into a target bacterial cell, wherein the DNA is capable of conjugal transfer into the target cell when in the presence of the component.

[0312] Thus, the DNA is capable of conjugal transfer into the target cell when the DNA is in the vector cell.

[0313] 25. The DNA of embodiment 24, wherein the DNA is according to any one of embodiments 1 to 23.

[0314] 26. The DNA of embodiment 24 or 25, wherein the cell is according to any one of embodiments 1 to 23.

[0315] 27. A method for enhancing growth or weight of an animal subject (e.g., a chicken), wherein the method comprises administering to a microbiome of the subject a plurality of the vector cells according to any one of embodiments 1-23, wherein the microbiome comprises target cells, and the first DNA is transferred from the vector cells into the target cells for expression therein to produce an antibacterial agent, thereby killing or reducing growth or proliferation of target cells (e.g., Salmonella cells) in the subject.

[0316] In an alternative, the administration is to a human for enhancing growth or weight of the human.

[0317] Optionally, the enhancing is not a medical therapy. Optionally, the enhancing is a medical therapy.

[0318] 28. A method for enhancing growth or weight of a plant (e.g., a tomato plant), wherein the method comprises administering to a microbiome of the plant a plurality of the vector cells according to any one of embodiments 1-23, wherein the microbiome comprises target cells, and the first DNA is transferred from the vector cells into the target cells for expression therein to produce an antibacterial agent, thereby killing or reducing growth or proliferation of target cells (e.g., Pseudomonas cells) in the plant.

[0319] The plant can be any plant disclosed herein. For example, the plant herein in any configuration or embodiment of the application is selected from the group consisting of a tomato plant, a potato plant, a wheat plant, a maize plant, a Zea mays plant, an apple tree, a legume-producing plant, a pea plant, a beetroot plant, a stone fruit plant, a barley plant, a hops plant, and a grass. For example, the plant is a tree, e.g., a palm, a yew, a pine, an oak, or a broadleaf tree. For example, the plant is a plant that produces a fruit selected from the group consisting of a strawberry, a raspberry, a blackberry, a red currant, a kiwifruit, a banana, an apple, an apricot, an avocado, a cherry, an orange, a clementine, a tangerine, a grapefruit, a plum, a date, a fig, a lime, a lemon, a melon, a mango, a pear, an olive, or a grape. Optionally, the plant is a dicot. Optionally, the plant is a flowering plant. Optionally, the plant is a monocot.

[0320] In any configuration, embodiment, or example herein, the target bacteria is a Pseudomonas syringae bacterium (e.g., comprised by the plant). Pseudomonas syringae pv. syringae Pseudomonas syringae pv. syringae ) is a common plant-associated bacterium that causes both monocot and dicot plant diseases worldwide. In one example, the target bacteria is a Pseudomonas syringae bacterium of a pathovar selected from the group consisting of: P. s. pv. aceris 、 P. s. pv. aptata 、 P. s.pv. atrofaciens 、 P. s. pv. dysoxylis 、 P. s. pv. japonica , Pseudomonas syringae pv. atalantae (PstA), Pseudomonas syringae pv. aurantifolia (PsaA), Pseudomonas syringae pv. avellanae (PsaAv), Pseudomonas syringae pv. coronafaciens (Psc), Pseudomonas syringae pv. garcae (Psg), Pseudomonas syringae pv. herculana (Psh), Pseudomonas syringae pv. ligulana (Psl), Pseudomonas syringae pv. morsprunorum (Psm), Pseudomonas syringae pv. phaseolicola (Psp), Pseudomonas syringae pv. syringae (Pss), and Pseudomonas syringae pv. zinniae (Psz). P. s. pv. lapsa , P. s. pv. panici , P. s. pv. papulans , P. s. pv. pisi , P. s. pv. morsprunorum .

[0321] • P. s. pv. aceris attacks Acer species.

[0322] • Pseudomonas syringae pv. actinidiae (PsaA) attacks Actinidia deliciosa. P. s. pv. actinidiae • Pseudomonas syringae pv. aesculi (PsaA) attacks Aesculus hippocastanum, causing canker disease.

[0323] • Pseudomonas syringae pv. betae (PsaA) attacks Beta vulgaris. P. s. pv. aesculi • Pseudomonas syringae pv. blakiana (PsaA) attacks Triticum aestivum.

[0324] • Pseudomonas syringae pv. coriaria (PsaA) attacks Triticum aestivum. P. s. pv. aptata • Pseudomonas syringae pv. garcae (Psg) attacks Triticum aestivum.

[0325] • Pseudomonas syringae pv. herculana (Psh) attacks Triticum aestivum.

[0326] • P. s. pv. dysoxylis attacks Dysoxylum spectabile.

[0327] • P. s. pv. japonica attacks Hordeum vulgare.

[0328] • Pseudomonas syringae pv. atalantae (PstA) attacks Triticum aestivum.

[0329] • P. syringae pv. stizense attacks Panicum grass species.

[0330] • P. syringae pv. herculaneum attacks Malus sylvestris species.

[0331] • P. syringae pv. phaseolicola causes halo blight of beans. P. s. pv. phaseolicola • P. syringae pv. lachrymans attacks Pisum sativum.

[0332] • P. syringae pv. syringae attacks Syringa, Prunus, and Phaseolus species.

[0333] • P. syringae pv. glycinea attacks Glycine max, causing bacterial blight of soybeans.

[0334] • P. syringae pv. atrosepticum attacks Malus sylvestris. P. s. pv. glycinea • P. syringae pv. atrosepticum attacks Malus sylvestris.

[0335] In one example, the target bacteria are P. syringae of a serotype recited in the bullet points in the immediately preceding paragraph, and the bacteria are comprised by a plant recited in the same bullet points.

[0336] In one example, the weight is dry weight. For example, the method is used to increase dry weight (e.g., within 1 or 2 weeks of the administration). Optionally, the increase is at least a 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% increase compared to a control plant of the same species or line for which the carrier cells were administered, wherein all plants are maintained under the same environmental conditions. For example, such an increase is within 1, 2, 3, 4, 5, 6, or 8 weeks after the first administration of carrier cells. In one example, the method is used to increase dry weight of leaves and / or fruit of a plant, e.g., a tomato plant.

[0337] In one example, the weight is wet weight. For example, the method is used to increase wet weight (e.g., within 1 or 2 weeks of the administration). Optionally, the increase is at least a 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% increase compared to a control plant of the same species or line for which no administration of the carrier cell was performed, wherein all plants are maintained under the same environmental conditions. For example, such increase is within 1, 2, 3, 4, 5, 6, or 8 weeks after the first administration of the carrier cell. In one example, the method is used to increase the dry weight of leaves and / or fruits of a plant, e.g., a tomato plant.

[0338] 29. The method of embodiment 28, wherein the microbiota is comprised by a leaf, main stem, root, or stem of the plant.

[0339] In one example, in any configuration or embodiment herein, the target bacteria (or target cell) is comprised by a microbiota of a plant. In one example, the microbiota is comprised by a leaf. In one example, the microbiota is comprised by xylem. In one example, the microbiota is comprised by phloem. In one example, the microbiota is comprised by a root. In one example, the microbiota is comprised by a tuber. In one example, the microbiota is comprised by a bulb. In one example, the microbiota is comprised by a seed. In one example, the microbiota is comprised by an exotesta, exocarp, mesocarp, or endocarp. In one example, the microbiota is comprised by a fruit, e.g., a simple fruit; an aggregate fruit; or a multiple fruit. In one example, the microbiota is comprised by a seed or embryo, e.g., by a seed coat; a seed leaf; a cotyledon; or a radicle. In one example, the microbiota is comprised by a flower, e.g., by a floral stem; a sepal; a petal; a stamen; a filament; an anther; or a pistil. In one example, the microbiota is comprised by a root; e.g., a taproot system or a fibrous root system. In one example, the microbiota is comprised by one or more leaves, e.g., by a leaf blade; a petiole; or a stipule. In one example, the microbiota is comprised by a stem, e.g., by bark; epidermis; phloem; cambium; xylem; or pith.

[0340] 30. A method for reducing a biofilm comprised by or on a surface of a subject, wherein the biofilm comprises a target cell (e.g., a Pseudomonas cell), wherein the method comprises administering a plurality of the carrier cells according to any one of embodiments 1 to 23 to the biofilm, wherein the first DNA is transferred from the carrier cell into the target cell for expression therein to produce an antibacterial agent, thereby killing or reducing growth or proliferation of the target cell in the biofilm.

[0341] In one example, "reducing a biofilm" includes reducing the coverage area of the biofilm. In one example, "reducing a biofilm" includes reducing the proliferation of the biofilm. In one example, "reducing a biofilm" includes reducing the durability of the biofilm. In one example, "reducing a biofilm" includes reducing the spread of the biofilm (e.g., in or on a subject, e.g., to an environment containing the subject).

[0342] 31. The method of embodiment 30, wherein the subject is a human or an animal.

[0343] For example, the biofilm is comprised by the lungs of the subject, e.g., wherein the target cells are Pseudomonas (e.g., Pseudomonas aeruginosa) cells. This can be useful in cases where the subject is a human having a lung disease or condition, e.g., pneumonia or cystic fibrosis.

[0344] For example, the biofilm is comprised by an animal or human organ disclosed herein. For example, the biofilm is comprised by the microbiota of a human or animal disclosed herein.

[0345] 32. The method of embodiment 30, wherein the subject is a plant (e.g., any plant disclosed herein).

[0346] Optionally, in the present embodiment, the target cells are Pseudomonas syringae cells.

[0347] 33. The method of embodiment 31 or 32, wherein the biofilm is comprised by a leaf, main stem, root, or stem of the plant.

[0348] In one example, in any configuration or embodiment herein, the target bacteria (or target cells) are comprised by a biofilm of a plant. In one example, the biofilm is comprised by a leaf. In one example, the biofilm is comprised by xylem. In one example, the biofilm is comprised by phloem. In one example, the biofilm is comprised by a root. In one example, the biofilm is comprised by a tuber. In one example, the biofilm is comprised by a bulb. In one example, the biofilm is comprised by a seed. In one example, the biofilm is comprised by an exotesta, exocarp, mesocarp, or endocarp. In one example, the biofilm is comprised by a fruit, e.g., a simple fruit; an aggregate fruit; or a multiple fruit. In one example, the biofilm is comprised by a seed or embryo, e.g., by a seed coat; a seed leaf; a cotyledon; or a radicle. In one example, the biofilm is comprised by a flower, e.g., by a floral stem; a sepal; a petal; a stamen; a filament; an anther; or a pistil. In one example, the biofilm is comprised by a root; e.g., a taproot system or a fibrous root system. In one example, the biofilm is comprised by one or more leaves, e.g., by a leaf blade; a petiole; or a stipule. In one example, the biofilm is comprised by a stem, e.g., by bark; a cuticle; phloem; a cambium; xylem; or pith.

[0349] 34. The method of any one of (e.g., 30) embodiments 30 to 33, wherein the surface is an ex vivo surface, such as a surface comprised by a domestic or industrial instrument or container.

[0350] 35. A method of replicating a first DNA to produce multiple copies of the DNA, the method comprising culturing a plurality of vector bacterial cells according to any one of embodiments 1 to 23, wherein the first DNA replicates in the cells.

[0351] Optionally, the method further comprises isolating the first DNA after the culturing. The skilled person is familiar with techniques and conditions that can be used to culture cells.

[0352] 36. The method of embodiment 35, comprising

[0353] (a) obtaining a sample of the cultured vector cells;

[0354] (b) contacting the sample of vector cells with a plurality of target bacterial cells to allow conjugation between the vector cells and the target cells; and

[0355] (c) allowing copies of the first DNA to be transferred from the vector cells to the target cells by conjugation, wherein an antibacterial agent is provided in the target cells and kills the target cells or reduces growth or proliferation of the target cells;

[0356] (d) wherein the first DNA is not replicable in the target cells.

[0357] For example, the first DNA is not substantially replicable in the target cells, such as less than 10, 5, 4, 3, 2 or 1% replication).

[0358] The first DNA is not (or not substantially) replicable in the target cells due to the absence of the first factor or a sequence encoding it in the target cells.

[0359] 37. A method of killing or reducing growth or proliferation of a plurality of target bacterial cells, the method comprising

[0360] (a) obtaining a sample of vector cells according to any one of embodiments 1 to 23 or obtainable by the method of embodiment 35;

[0361] (b) contacting the sample of vector cells with a plurality of target bacterial cells to allow conjugation between the vector cells and the target cells; and

[0362] (c) allowing copies of the first DNA to be transferred from the vector cells to the target cells by conjugation, wherein an antibacterial agent is provided in the target cells and kills the target cells or reduces growth or proliferation of the target cells;

[0363] (d) wherein the first DNA is not (or not substantially) replicable in the target cell.

[0364] 38. The method of embodiment 36 or 37, wherein the target cells are comprised by a biofilm, e.g., a biofilm as disclosed herein.

[0365] 39. A method for suppressing the action of an antibacterial agent in an environment, wherein the agent is toxic to a target bacterial cell, the method comprising performing the method of embodiment 37 or 38, and the agent is the agent recited in embodiment.

[0366] 40. The method of embodiment 39, wherein the environment is comprised by a human or animal subject, and the target cells are comprised by a biofilm of the subject, wherein the method comprises administering the sample of carrier cells to the biofilm of the subject, wherein the carrier cells are contacted with the target cells in step (b), wherein the method is a suppression method for treating or preventing a disease or condition mediated by the target cells in the subject.

[0367] 41. The carrier cell, DNA, or method of any preceding embodiment, wherein the target bacteria is a Salmonella, Pseudomonas, Escherichia ( Escherichia ), Klebsiella ( Klebsiella ), Campylobacter, Helicobacter ( Helicobacter ), Acinetobacter ( Acinetobacter ), Enterobacteriaceae, Clostridium, Staphylococcus, or Streptococcus bacteria.

[0368] 42. The carrier cell, DNA, or method of any preceding embodiment, wherein the target bacteria is an Enterica Salmonella bacteria.

[0369] For example, the target bacteria is selected from the group consisting of Enterica Salmonella enterica subspecies typhimurium, enteritidis, villosus, montevideo, hadar, and bongia serotypes.

[0370] 43. The carrier cell, DNA, or method of any one of embodiments 1 to 41, wherein the target bacteria is a Pseudomonas (e.g., P. syringae or P. aeruginosa) bacteria.

[0371] 44. The carrier cell or method of any one of embodiments 1 to 41, wherein the target bacteria is an Escherichia coli bacteria.

[0372] Optionally, the target bacteria is an enterohemorrhagic E. coli (EHEC), E. coli serotype 0157:H7, or a Shiga toxin-producing E. coli (STEC). In one example, the target bacteria is selected from the group consisting of

[0373] • Shiga toxin-producing E. coli (STEC) (STEC can also be referred to as Verotoxin-producing E. coli (VTEC);

[0374] • Enterohemorrhagic E. coli (EHEC) (this pathotype is the most commonly heard in the news in relation to foodborne outbreaks);

[0375] • Enterotoxigenic E. coli (ETEC);

[0376] • Enteropathogenic E. coli (EPEC);

[0377] • Enteraggregative E. coli (EAEC);

[0378] • Enteroinvasive E. coli (EIEC); and

[0379] • Diffuse adherent E. coli (DAEC).

[0380] Enterohemorrhagic E. coli (EHEC) serotype O157:H7 is a human pathogen that causes outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Conventional antimicrobials trigger the SOS response in EHEC, which promotes the release of potent Shiga toxins that cause most of the morbidity and mortality associated with EHEC infection. Cattle are a natural reservoir for EHEC, and approximately 75% of EHEC outbreaks are linked to the consumption of contaminated bovine-derived products. EHEC causes disease in humans, but is asymptomatic in adult ruminants. Characteristics of E. coli serotype O157:H7 (EHEC) infection include abdominal cramping and bloody diarrhea, and the life-threatening complication hemolytic uremic syndrome (HUS). There is a current need for a treatment for EHEC infection (Goldwater and Bettelheim, 2012). The use of conventional antibiotics exacerbates Shiga toxin-mediated cytotoxicity. In an epidemiological study by the Centers for Disease Control and Prevention, patients with EHEC enteritis treated with antibiotics had a higher risk of developing HUS (Slutsker et al., 1998). Additional studies support antibiotic contraindication in EHEC infection; children who received antibiotic therapy for hemorrhagic colitis associated with EHEC had an increased chance of developing HUS (Wong et al., 2000; Zimmerhackl, 2000; Safdar et al., 2002; Tarr et al., 2005). Conventional antibiotics promote Shiga toxin production by enhancing stx replication and expression of genes that stxThe genes are encoded within the chromosome-integrated lambda phage genome. The methods of some configurations of the invention rely on nuclease cleavage. Stx Induction also promotes phage-mediated lysis of the EHEC extracellular capsule, allowing release and dissemination of Shiga toxin into the environment (Karch et al., 1999; Matsushiro et al., 1999; Wagner et al., 2002). Thus, advantageously, these configurations of the invention provide an alternative means for treating EHEC in human and animal subjects. This is exemplified below with the surprising results regarding the speed and duration of anti-EHEC action produced by nuclease action (as opposed to conventional antibiotic action).

[0381] In one example, the subject (e.g., human or animal) has, or is at risk of, hemolytic uremic syndrome (HUS), e.g., the subject has an E. coli infection, e.g., an EHEC E. coli infection.

[0382] 45. A pharmaceutical composition, a livestock growth promoting composition, a soil amendment, a herbicide, a plant fertilizer, a food or food ingredient sterilizing composition, a dental composition, a personal hygiene composition, or a disinfectant composition (e.g., for domestic or industrial use) comprising a plurality of the carrier cells according to any one of embodiments 1 to 23.

[0383] Herein, a carrier cell is, for example, a probiotic cell for administration to a human or animal subject. For example, the carrier cell is commensal in the microbiome (e.g., gut or blood microbiome) of a human or animal subject, wherein the carrier is for administration to the subject. In one example, the carrier cell is a bacterial cell (and optionally, the target cell is a bacterial cell). In one example, the carrier cell is an archaeal cell (and optionally, the target cell is an archaeal cell).

[0384] Optionally, the carrier cell is a Gram-positive bacterial cell, and the target cell is a Gram-positive bacterial cell.

[0385] Optionally, the carrier cell is a Gram-positive bacterial cell, and the target cell is a Gram-negative bacterial cell.

[0386] Optionally, the carrier cell is a Gram-negative bacterial cell, and the target cell is a Gram-positive bacterial cell.

[0387] Optionally, the carrier cell is a Gram-negative bacterial cell, and the target cell is a Gram-negative bacterial cell.

[0388] Optionally, the carrier cell is a Bacillus bacterial cell, and the target cell is a Gram-positive bacterial cell.

[0389] Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a Gram-negative bacterial cell.

[0390] Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is a Salmonella bacterial cell.

[0391] Optionally, the carrier cell is a Bacillus bacterial cell and the target cell is an Escherichia coli bacterial cell.

[0392] Optionally, the carrier cell is an Escherichia coli bacterial cell and the target cell is a Pseudomonas bacterial cell.

[0393] Optionally, the carrier cell is an Escherichia coli bacterial cell and the target cell is a Gram-positive bacterial cell.

[0394] Optionally, the carrier cell is an Escherichia coli bacterial cell and the target cell is a Gram-negative bacterial cell.

[0395] Optionally, the carrier cell is an Escherichia coli bacterial cell and the target cell is a Salmonella bacterial cell.

[0396] Optionally, the carrier cell is an Escherichia coli bacterial cell and the target cell is an Escherichia coli bacterial cell.

[0397] Optionally, the carrier cell is an Escherichia coli bacterial cell and the target cell is a Pseudomonas bacterial cell.

[0398] The Bacillus cell herein is optionally a Bacillus subtilis cell.

[0399] Optionally, the carrier cell is a probiotic or commensal Escherichia coli bacterial cell for administration to a human or animal subject. Optionally, the carrier cell is a probiotic or commensal Bacillus bacterial cell for administration to a human or animal subject.

[0400] In this context, optionally, the first DNA is comprised by a plasmid, e.g. a closed circular DNA.

[0401] In one embodiment, in one instance, the first DNA is a dsDNA. In one embodiment, in one instance, the first DNA is a ssDNA.

[0402] In an alternative configuration, the first DNA is instead a first RNA.

[0403] Optionally, the target cell is a Salmonella cell (e.g. where the carrier cell is an Escherichia coli cell), e.g. Salmonella enterica enterica subsp., e.g. Salmonella enterica enterica subsp. typhimurium, enteritidis, wilseria, montevideo, hadar or bongia serovar.

[0404] For example, the target bacteria are selected from Salmonella enterica; Salmonella typhimurium; Pseudomonas aeruginosa; Escherichia coli; Klebsiella pneumoniae; Campylobacter jejuni; Helicobacter pylori H pylori ); Acinetobacter baumannii A baumanii ); Clostridium difficile C difficile ); Staphylococcus aureus S aureus ); Streptococcus pyogenes S pyogenes ); or Streptococcus thermophilus S thermophilus ).

[0405] In one example, the target cells are cells of a species that causes a hospital infection in a human.

[0406] Optionally, the target cells are comprised by a microbiome of an animal (e.g., a poultry animal (e.g., a chicken), a pig, a cow, a fish (e.g., a catfish or a salmon), or a shellfish (e.g., a prawn or a lobster)). Optionally, the microbiome is a gut microbiome. For example, the target cells are Salmonella cells comprised by a chicken gut biofilm. For example, the target cells are Salmonella cells comprised by an ex vivo chicken gut biofilm sample.

[0407] In one embodiment, the first DNA comprises a bacterial oriV and / or oriT. In one embodiment, the first DNA is comprised by a plasmid, wherein the plasmid comprises an oriV and / or an oriT.

[0408] The first factor can be a protein or an RNA. For example, the first factor is pir or trfA In one example, the first factor is operable with the oriV comprised by the first DNA for replication thereof.

[0409] In one embodiment, the first DNA is comprised by a plasmid, wherein the plasmid comprises an oriV and does not encode any replication protein operable with the oriV to initiate replication of the plasmid (e.g., pir or trfA ).

[0410] In one embodiment, the first DNA lacks a component required for conjugative transfer of the first DNA into a target bacterial cell. Optionally, the component is a protein. Optionally, the component is an RNA. Optionally, the component is a tra1 component. Optionally, the component is a tra2 component. In one example, the vector cell comprises the component, wherein the component is comprised or encoded by a vector cell chromosome, a second DNA in the vector cell, or a third DNA. Preferably, the component is comprised or encoded by the vector cell chromosome.

[0411] Optionally, the component required for conjugative transfer is a component of an RP4 plasmid, e.g., a component of an RP4 tra module (e.g., a tra1 or tra2 module).

[0412] Optionally, the component required for conjugative transfer is a RK2 plasmid component, e.g. a component of a RK2 tra module (e.g. a tra1 or tra2 module).

[0413] Optionally, the component required for conjugative transfer is a R6K plasmid component, e.g. a component of a R6K tra module (e.g. a tra1 or tra2 module).

[0414] Optionally, the first DNA is comprised by a plasmid which does not comprise an antibiotic resistance marker gene and / or a plasmid addiction system gene. As explained in the Examples section, in the rare case that an IncP equivalent plasmid is present in the target cell which would provide the trfA (or other component required for conjugation, wherein said component is not encoded by the plasmid comprising the first DNA), both plasmids would compete for the available TrfA (or other component), resulting in loss of one of the plasmids, and thus would be lost from any progeny target cell very soon.

[0415] Optionally, the plasmid comprising the first DNA further comprises an anti-restriction gene, which encodes a product for inhibiting a restriction enzyme (e.g. a type I restriction enzyme) of the target cell, such as an anti-restriction gene which inhibits a type I restriction enzyme (e.g. the anti-restriction gene product is ocr of T7, klcA, ard or ardB of RK2).

[0416] Additionally or alternatively, the plasmid comprises a gene encoding an essential component of a type IV secretion system, wherein the chromosome of the carrier cell comprises a gene encoding the remaining part of the secretion system, wherein said essential component is required for the conjugative transfer of the plasmid from the carrier cell to the target cell. For example, in addition to the tra1 or tra2 gene encoding the essential component, the chromosome comprises all remaining parts of the traKLM, traJXIHGF genes of RK2 tra1, and all remaining parts of the trbBCEFGHJL genes of RK2 tra2 (preferably when the plasmid is of RK2 type). In an alternative example, in addition to the gene encoding the essential component, the chromosome comprises all remaining parts of the RK6 homologues of the traKLM, traJXIHGF genes of RK2 tra1, and all remaining parts of the RK6 homologues of the trbBCEFGHJL genes of RK2 tra2 (preferably when the plasmid is of RK6 type). In an alternative example, in addition to the gene encoding the essential component, the chromosome comprises all remaining parts of the RP4 homologues of the traKLM, traJXIHGF genes of RK2 tra1, and all remaining parts of the RP4 homologues of the trbBCEFGHJL genes of RK2 tra2 (preferably when the plasmid is of RP4 type).

[0417] For example, in addition to the tra 1 or tra 2 gene encoding the essential component, the chromosome comprises all of the remainder of the RK2 plasmid DNA segment from traF to traM, and all of the remainder of the RK2 plasmid DNA segment from trbB to trK (preferably, when the plasmid is an RK2-type plasmid). For example, in addition to the tra 1 or tra 2 gene encoding the essential component, the chromosome comprises all of the remainder of the RK2 plasmid gene from traF to traM, and all of the remainder of the RK2 plasmid gene from trbB to trK (preferably, when the plasmid is an RK2-type plasmid). For example, the chromosome comprises (in the order 5' to 3', or 3' to 5') the following RK2 tra 1 genes: traFGHIXJKLM, or traKLM, or traJXIHGF. For example, the chromosome comprises (in the order 5' to 3', or 3' to 5') the following RK2 tra 2 genes: trbBCDEFGHIJKL or trbBCEFGHJL.

[0418] For example, in addition to the gene encoding the essential component, the chromosome comprises all of the remainder of the RK6 plasmid DNA segment from the homolog of RK2 traF to the homolog of RK2 traM, and all of the remainder of the RK6 plasmid DNA segment from the homolog of RK2 trbB to the homolog of RK2 trK (preferably, when the plasmid is an RK6-type plasmid). For example, in addition to the gene encoding the essential component, the chromosome comprises all of the remainder of the RK6 plasmid gene from the homolog of RK2 traF to the homolog of RK2 traM, and all of the remainder of the RK6 plasmid gene from the homolog of RK2 trbB to the homolog of RK2 trK (preferably, when the plasmid is an RK6-type plasmid). For example, the chromosome comprises (in the order 5' to 3', or 3' to 5') the RK6 homolog of the following RK2 tra 1 genes: traFGHIXJKLM, or traKLM, or traJXIHGF. For example, the chromosome comprises (in the order 5' to 3', or 3' to 5') the RK6 homolog of the following RK2 tra 2 genes: trbBCDEFGHIJKL or trbBCEFGHJL.

[0419] For example, in addition to the genes encoding the essential components, the chromosome comprises all of the remainder of the DNA segment of the RP4 plasmid from the homolog of RK2 traF to the homolog of RK2 traM, and all of the remainder of the DNA segment of the RP4 plasmid from the homolog of RK2 trbB to the homolog of RK2 trK (preferably, when the plasmid is an RP4-type plasmid). For example, in addition to the genes encoding the essential components, the chromosome comprises all of the remainder of the RP4 plasmid genes from the homolog of RK2 traF to the homolog of RK2 traM, and all of the remainder of the RP4 plasmid genes from the homolog of RK2 trbB to the homolog of RK2 trK (preferably, when the plasmid is an RP4-type plasmid). For example, the chromosome comprises (in the order 5' to 3', or 3' to 5') the RP4 homologs of the following RK2 tral genes: traFGHIXJKLM, or traKLM, or traJXIHGF. For example, the chromosome comprises (in the order 5' to 3', or 3' to 5') the RP4 homologs of the following RK2 tra2 genes: trbBCDEFGHIJKL or trbBCEFGHJL.

[0420] Preferably, the second DNA or the chromosome comprising the second DNA lacks an oriT. Preferably, the third DNA or the chromosome comprising the third DNA lacks an oriT.

[0421] For example, the oriT is only comprised by the first DNA (or the plasmid comprising it) and not by any other DNA in the vector cell. Thus, transfer is limited to the first DNA or its plasmid.

[0422] The first DNA in all embodiments and configurations can preferably not be comprised by an out-of-control replicating plasmid. Naturally occurring plasmids exist within a host cell at a characteristic concentration (referred to herein as the “copy number” of the particular plasmid). Mutations that disrupt the elements of control lead to an over-replicating phenotype, which manifests as an increase in the copy number of the plasmid (“copy-up” phenotype). In the extreme case of a copy-up mutation, the plasmid replicates completely uncontrolled due to the loss of copy control mechanisms. This is referred to as “out-of-control plasmid replication” or simply “out-of-control replication”, and a plasmid that participates in such out-of-control replication is an “out-of-control replicating plasmid”.

[0423] In one example, the application relates to a composition comprising a plurality of vector cells of the application (e.g., wherein copies of the first DNA are contained by separate plasmids). Optionally, all of the vector cells comprise identical said first DNA and second DNA. Optionally, the plurality of vector cells comprises a first subpopulation of vector cells (first cells) and a second subpopulation of vector cells (second cells), wherein the first cells comprise identical first DNA, and the second cells comprise identical first DNA (which is different from the first DNA of the first cells). For example, the preceding DNA comprises a different NSI than the NSI comprised by the other DNA. For example, the first DNA encodes a first guide RNA or crRNA, and the second DNA encodes a second guide RNA or crRNA, wherein the first guide RNA / crRNA is capable of hybridizing to a first protospacer sequence in a first target cell; and the second guide RNA / crRNA is capable of hybridizing to a second protospacer sequence in a second target cell, wherein the protospacer sequences are different. Optionally, the first target cell is different from the second target cell. Optionally, the first target cell has the same species or strain as the second target cell. Alternatively, the first target cell has a different species or strain than the second target cell (in this way providing a cocktail of vector cells, e.g., for administration to a human or animal or plant, to target and kill a plurality of target cells of different species or strains).

[0424] In one example, the first or each first DNA comprises a plurality of (e.g., first and second) NSIs, wherein the first NSI is different from the second NSI (e.g., they encode different proteins or RNAs, e.g., different guide RNAs or crRNAs). In one example, the first or each first DNA comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different types of NSIs. In one example, the first or each first DNA comprises NSIs that encode 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different guide RNAs. In one example, the first or each first DNA comprises NSIs that encode 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different crRNAs. In one example, the first or each first DNA comprises NSIs that encode at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different guide RNAs. In one example, the first or each first DNA comprises NSIs that encode at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 different crRNAs.

[0425] Optionally, the composition is comprised by a liquid (e.g., an aqueous liquid or in water), the composition comprising the carrier cells in an amount of 1 x 10 3 to 1 x 10 10 (e.g., 1 x 10 4 to 1 x 10 10 ; 1 x 10 4 to 1 x 10 9 ; 1 x 10 4 to 1 x 10 8 ; 1 x 10 4 to 1 x 10 7 ; 1 x 10 3 to 1 x 10 10 ; 1 x 10 3 to 1 x 10 9 ; 1 x 10 3 to 1 x 10 8 ; 1 x 10 3 to 1 x 10 7 ; 1 x 10 5 to 1 x 10 10 ; 1 x 105 to 1 x 10 9 ; 1 x 10 5 to 1 x 10 8 ; 1 x 10 5 to 1 x 10 7 ; 1 x 10 6 to 1 x 10 10 ; 1 x 10 6 to 1 x 10 9 ; 1 x 10 6 to 1 x 10 8 ; or 1 x 10 6 to 1 x 10 7 ) cfu / ml. For example, the liquid is a beverage, e.g., for human or animal consumption. For example, the beverage is a livestock beverage, e.g., a poultry beverage (i.e., a beverage for consumption by poultry, e.g., chickens).

[0426] In one example, the composition is a diet (e.g., a diet supplement) composition for consumption by a human or an animal. In one example, the composition is a weight loss composition for consumption by a human or an animal. In one example, the composition is a growth promotion composition for consumption by a human or an animal. In one example, the composition is a fitness composition for consumption by a human. In one example, the composition is a probiotic composition for consumption by a human or an animal. In one example, the composition is a biocide composition for consumption by a human or an animal. In one example, the composition is a pesticide composition for consumption by a human or an animal. In one example, the composition is a zoonosis control composition for consumption by an animal.

[0427] In one example, the composition comprises, in addition to the carrier cells, a vitamin. In one example, the composition comprises, in addition to the carrier cells, vitamin A, B (e.g., B12), C, D, E, and / or K. In one example, the composition comprises, in addition to the carrier cells, a lipid. In one example, the composition comprises, in addition to the carrier cells, a carbohydrate. In one example, the composition comprises, in addition to the carrier cells, a protein and / or an amino acid. In one example, the composition comprises, in addition to the carrier cells, a mineral. In one example, the composition comprises, in addition to the carrier cells, a metal ion (e.g., Mg 2+ , Cu 2+ , and / or Zn 2+ ). In one example, the composition comprises sodium ions, potassium ions, magnesium ions, calcium ions, manganese ions, iron ions, cobalt ions, copper ions, zinc ions, and / or molybdenum ions.

[0428] In one example, the composition is a plant fertilizer composition. In one example, the composition is a herbicide. In one example, the composition is a pesticide composition for application to a plant.

[0429] In any embodiment or example, as appropriate: the plant is, for example, a crop plant. The plant is, for example, wheat. The plant is, for example, corn. The plant is, for example, maize. The plant is, for example, a fruiting plant. The plant is, for example, a vegetable plant. The plant is, for example, a tomato plant. The plant is, for example, a potato plant. The plant is, for example, a grass plant. The plant is, for example, a flowering plant. The plant is, for example, a tree. The plant is, for example, a shrub.

[0430] In one example, the composition is for environmental application, wherein the environment is an outdoor environment (e.g., application to a field or waterway or reservoir).

[0431] In one example, the composition is comprised by a food or food ingredient (e.g., for human or animal consumption). In one example, the composition is comprised by a beverage or beverage ingredient (e.g., for human or animal consumption).

[0432] In one example, the target cell is a human biomembrane cell, for example, wherein the biomembrane is an intestinal, skin, lung, eye, nose, ear, gastrointestinal tract (GI tract), stomach, hair, kidney, urethra, bronchiole, oral cavity, mouth, liver, heart, anus, rectum, bladder, bowel, intestine, penis, vagina, or scrotum biomembrane. In one example, the target cell is an animal biomembrane cell, for example, wherein the biomembrane is an intestinal, skin, lung, eye, nose, ear, gastrointestinal tract (GI tract), cecum, jejunum, ileum, colon, stomach, feather, hair, scale, kidney, urethra, bronchiole, oral cavity, mouth, liver, spleen, heart, anus, rectum, bladder, bowel, intestine, penis, vagina, or scrotum biomembrane. For example, the biomembrane is a bird (e.g., chicken) cecum biomembrane. For example, the biomembrane is a bird (e.g., chicken) gastrointestinal tract (GI tract), cecum, jejunum, ileum, colon, or stomach biomembrane.

[0433] In one example, any method herein is ex vivo. In one example, a method herein is in vivo. In one example, a method herein is in vitro. In one example, a method herein is performed in an environment, for example, in a home (e.g., house), industrial (e.g., factory), or agricultural environment (e.g., field). In one example, a method herein is performed in or on a container; or on a surface.

[0434] In one example, the NSI (or RNA product thereof) is capable of recombining with a target cell chromosome or episome comprised by the target cell to modify the chromosome or episome. Optionally, this is performed in a method in which the chromosome or episome is cleaved (e.g., using a guide nuclease such as Cas, TALEN, zinc finger nuclease, or meganuclease, at a predetermined site), and simultaneously or sequentially, by conjugation with a vector cell, a first DNA is introduced into the target cell, and the NSI or sequence thereof is inserted into the chromosome or episome at or near the cleavage site.

[0435] In one example, the first DNA comprises one or more components of a CRISPR / Cas system operable to perform protospacer cleavage in the target cell (e.g., wherein the protospacer comprises 10-20, 10-30, 10-40, 10-100, 12-15, or 12-20 contiguous nucleotides capable of hybridizing in the target cell to a crRNA or gRNA encoded by the NSI).

[0436] For example, the system is a Type I, II, III, IV, or V CRISPR / Cas system.

[0437] In one example, the NSI encodes Cas9 (and optionally a second, different Cas, such as Cas3, Cas9, Cpfl, Casl3a, Casl3b, or CaslO). In one example, the NSI encodes Cas3 (and optionally a second, different Cas, such as Cas3, Cas9, Cpfl, Casl3a, Casl3b, or CaslO). In one example, the NSI encodes a Cas selected from Cas3, Cas9, Cpfl, Casl3a, Casl3b, and CaslO. Additionally or alternatively, the first DNA (e.g., the NSI) encodes a guide RNA or crRNA or tracrRNA. For example, the guide RNA or crRNA or tracrRNA is homologous to the first Cas (i.e., operable within the target cell).

[0438] In one example, the Cas herein is Cas9. In one example, the Cas herein is Cas3. The Cas can be identical to a Cas encoded by the target bacteria.

[0439] In one embodiment, the presence of the NSI or its encoded protein or RNA in the target bacteria mediates target cell killing, or downregulation of target cell growth or reproduction. In one embodiment, the presence of the NSI or its encoded protein or RNA in the target bacteria mediates the shutdown, or downregulation, of expression of one or more RNAs or proteins encoded by the target cell genome.

[0440] In one embodiment, the presence of the NSI, or a protein or RNA encoded thereby, in the target bacteria mediates upregulation of growth or reproduction of the target cell. In one embodiment, the presence of the NSI, or a protein or RNA encoded thereby, in the target bacteria mediates turning on, or upregulation of, expression of one or more RNAs or proteins encoded by the genome of the target cell.

[0441] In one embodiment, the NSI encodes a component of a CRISPR / Cas system that is toxic to the target bacteria.

[0442] In one embodiment, the first DNA is comprised by a plasmid or a shuttle vector. In one embodiment, the second DNA is comprised by a vector (e.g., a plasmid or a shuttle vector), a helper phage (e.g., a helper phasmid), or is integrated in the genome of the host bacterial cell.

[0443] Optionally, the target cell lacks a functional endogenous CRISPR / Cas system prior to transfer of the first DNA (e.g., the first DNA comprising a component of an exogenous CRISPR / Cas system that is functional in and toxic to the target cell) thereto. One embodiment provides an antibacterial composition comprising a plurality of the vector cells of the application, wherein each target cell is optionally in accordance with this paragraph, for administration to a human or animal subject for medical use.

[0444] In one example, the composition of the application is a herbicide, a pesticide, an insecticide, a plant fertilizer, or a cleaning agent.

[0445] Optionally, the target bacteria herein are comprised by a microbiome of the subject, e.g., a gut microbiome. Alternatively, the microbiome is a skin, scalp, hair, eye, ear, mouth, throat, lung, blood, rectum, anus, vaginal, scrotal, penile, nasal, or tongue microbiome.

[0446] In one example, a drug is further administered to the subject (e.g., a human or animal) concurrently or sequentially with the administration of the vector cell. In one example, the drug is an antibiotic, an antibody, an immune checkpoint inhibitor (e.g., an anti-PD-1, anti-PD-L1, or anti-CTLA4 antibody), an adoptive cell therapy (e.g., a CAR-T therapy), or a vaccine.

[0447] In one embodiment, the NSI encodes a guide nuclease, e.g., a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease. Thus, the toxic agent can comprise a guide nuclease, e.g., a Cas nuclease, a TALEN, a zinc finger nuclease, or a meganuclease. Optionally, the NSI encodes a restriction nuclease capable of cleaving a chromosome of the target cell.

[0448] Optionally, the composition is a pharmaceutical composition in a medicament for practicing on a human or animal subject.

[0449] In one instance, the animal is livestock or a companion pet (e.g., a cow, pig, goat, sheep, horse, dog, cat, or rabbit). In one instance, the animal is an insect (an insect at any stage of its life cycle, such as an egg, larva, or pupa). In one instance, the animal is a protozoan. In one instance, the animal is a cephalopod.

[0450] Optionally, the composition is a herbicide, pesticide, food or beverage processing aid, food or beverage additive, petrochemical or fuel processing aid, water purifier, cosmetic additive, detergent additive, or environmental (e.g., soil) additive or cleaner.

[0451] The present invention also provides: a target bacterial cell or multiple target bacterial cells, each containing the first DNA.

[0452] For example, the carrier bacteria are Lactobacillus species (e.g., Lactobacillus reuteri). L reuteri ) or Lactococcus lactis ( L lactis (e.g., Escherichia coli, Bacillus spp., or Streptococcus spp. (e.g., Streptococcus thermophilus)). Usefully, the vector can provide protection of the first DNA from its surrounding environment. The vector can be used for oral administration or other routes, wherein the vector provides protection of the first DNA from stomach acid or other harsh environments in the subject. Furthermore, the vector can be formulated into beverages, such as probiotic beverages, such as adapted Yakult (trademark), Actimel (trademark), Kevita (trademark), Activia (trademark), Jarrow (trademark), or similar beverages for human consumption.

[0453] Optionally, the carrier cells or composition are used to administer to human or animal subjects for medical purposes, which includes killing target bacteria using a reagent or an expression product of an NSI, wherein said target bacteria mediate a disease or condition in the subject. In one instance, when the subject is a human, the subject is not an embryo. In one instance, the carrier cells are probiotics in the subject.

[0454] The present invention also provides a method for killing target bacterial cells in the environment, optionally wherein the method is not practiced on human or animal bodies, wherein the method includes exposing the environment to the carrier cells or composition of the present invention and allowing the expression of an NSI product in the target cells, wherein the target bacteria are killed in the presence of the product. For example, the product encodes a CRISPR / Cas system or a component thereof, such as the systems or components disclosed herein. Thus, the system may be able to recognize and cleave the anterior chromosomal spacer sequence of the target cells, thereby killing the target cells. Optionally, in a further step, the killed target cells are isolated.

[0455] The application also provides: use of a composition or cell of the application in the manufacture of an antibacterial agent that kills a target bacterium, for treating a disease or condition in a human or animal subject comprising the target bacterium.

[0456] Optionally, the environment is a microbiome of: soil; a plant, part (e.g. a leaf, fruit, vegetable or flower) or a part of a plant product (e.g. meat); water; a waterway; a fluid; a food or component thereof; a beverage or component thereof; a medical device; a cosmetic; a detergent; blood; a bodily fluid; a medical instrument; an industrial instrument; an oil rig; an oil chemical processing, storage or transport instrument; a vehicle or a shipping container.

[0457] Optionally, the environment is an ex vivo bodily fluid (e.g. urine, blood, a blood product, sweat, tears, sputum or saliva), bodily solid (e.g. faeces) or tissue of a human or animal subject to which the composition has been administered.

[0458] Optionally, the environment is an in vivo bodily fluid (e.g. urine, blood, a blood product, sweat, tears, sputum or saliva), bodily solid (e.g. faeces) or tissue of a human or animal subject to which the composition has been administered.

[0459] In one embodiment, the first DNA is comprised by a phagemid or a cloning vector (e.g. a shuttle vector, e.g. a pUC vector).

[0460] In one embodiment, the second DNA is comprised by a bacterial vector cell chromosome.

[0461] Optionally, the toxic agent comprises one or more components of a CRISPR / Cas system, e.g. a DNA sequence encoding one or more components of a type I cascade (e.g. CasA).

[0462] Optionally, the toxic agent comprises a DNA sequence encoding a guide nuclease, e.g. a Cas nuclease, a TALEN, a zinc finger nuclease or a meganuclease.

[0463] In one example, the carrier cells or compositions are contained by a medical container, such as a syringe, vial, IV bag, inhaler, eyedropper, or nebulizer. In one example, the carrier cells or compositions are contained by a sterile container. In one example, the carrier cells or compositions are contained by a medically compatible container. In one example, the carrier cells or compositions are contained by a fermentation vessel, such as a metal, glass, or plastic vessel. In one example, the carrier cells or compositions are contained by an agricultural instrument. In one example, the carrier cells or compositions are contained by a food production or processing instrument. In one example, the carrier cells or compositions are contained by a horticultural instrument. In one example, the carrier cells or compositions are contained by a farming instrument. In one example, the carrier cells or compositions are contained by a petrochemical recovery or processing instrument. In one example, the carrier cells or compositions are contained by a distillation instrument. In one example, the carrier cells or compositions are contained by a cell culture vessel (e.g., having a capacity of at least 50, 100, 1000, 10000, or 100000 liters). Additionally or alternatively, the target cells are contained by any of these instruments, etc.

[0464] In one example, the carrier cells or compositions are contained by a drug, such as in combination with an instruction or package label indicating that the drug is to be administered by oral, IV, subcutaneous, intranasal, intraocular, vaginal, topical, rectal, or inhalation administration to a human or animal subject. In one example, the carrier cells or compositions are contained by an oral drug formulation. In one example, the carrier cells or compositions are contained by an intranasal or intraocular drug formulation. In one example, the carrier cells or compositions are contained by a personal hygiene composition (e.g., shampoo, soap, or deodorant) or cosmetic formulation. In one example, the carrier cells or compositions are contained by a detergent formulation. In one example, the carrier cells or compositions are contained by a cleaning formulation, such as for cleaning a medical or industrial device or instrument. In one example, the carrier cells or compositions are contained by a food, food ingredient, or food processing aid. In one example, the carrier cells or compositions are contained by a beverage, beverage ingredient, or beverage processing aid. In one example, the carrier cells or compositions are contained by a medical bandage, fabric, cast, or swab. In one example, the carrier cells or compositions are contained by a herbicide or pesticide. In one example, the carrier cells or compositions are contained by an insecticide.

[0465] In one example, the CRISPR / Cas component is a component of a Type I CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a Type II CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a Type III CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a Type IV CRISPR / Cas system. In one example, the CRISPR / Cas component is a component of a Type V CRISPR / Cas system. In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding a Cas9 (e.g., S. pyogenes Cas9, S. aureus Cas9, or S. thermophilus Cas9). In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding a Cas3 (e.g., E. coli Cas3, C. difficile Cas3, or Salmonella Cas3). In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding a Cpf. In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding a CasX. In one example, the CRISPR / Cas component comprises a nucleotide sequence encoding a CasY.

[0466] In one example, each vector cell encodes a CRISPR / Cas component or a protein of interest from a nucleotide sequence (NSI) comprising a promoter operable in the target bacteria.

[0467] Optionally, the target bacteria is a Gram-negative bacteria (e.g., a spirillum or a vibrio). Optionally, the target bacteria is a Gram-positive bacteria. Optionally, the target bacteria is a mycoplasma, a chlamydiae, a spirochete, or a mycobacterium bacteria. Optionally, the target bacteria is a Streptococcus (e.g., S. pyogenes or S. thermophilus). Optionally, the target bacteria is a Staphylococcus (e.g., S. aureus, e.g., MRSA). Optionally, the target bacteria is an E. coli (e.g., O157:H7), e.g., where Cas is encoded by the vector or endogenous target cell Cas nuclease (e.g., Cas3) activity is de-repressed. Optionally, the target bacteria is a Pseudomonas (e.g., P. syringae or P. aeruginosa). Optionally, the target bacteria is a Vibrio (e.g., V. cholerae (e.g., O139) or V. vulnificus). Optionally, the target bacteria is a Neisseria (e.g., N. gonorrhoeae or N. meningitidis). cholerae ) (e.g., N. gonorrhoeae (e.g., O139) or V. vulnificus). vulnificus )) (e.g., N. gonorrhoeae (e.g., O139) or V. vulnificus). Neisseria ) (e.g., N. gonorrhoeae (e.g., O139) or V. vulnificus). gonnorrhoeae ) (e.g., N. gonorrhoeae (e.g., O139) or V. vulnificus). meningitidis)). Optionally, the target bacteria is Bordetella ( Bordetella ) (e.g. Bordetella pertussis ( pertussis )). Optionally, the target bacteria is Haemophilus ( Haemophilus ) (e.g. Haemophilus influenzae ( influenzae )). Optionally, the target bacteria is Shigella ( Shigella ) (e.g. Shigella dysenteriae ( dysenteriae )). Optionally, the target bacteria is Brucella ( Brucella ) (e.g. Brucella abortus ( abortus )). Optionally, the target bacteria is Francisella ( Francisella ) host. Optionally, the target bacteria is Xanthomonas ( Xanthomonas ). Optionally, the target bacteria is Agrobacterium ( Agrobacterium ). Optionally, the target bacteria is Erwinia ( Erwinia ). Optionally, the target bacteria is Legionella ( Legionella ) (e.g. Legionella pneumophila ( pneumophila )). Optionally, the target bacteria is Listeria (e.g. Listeria monocytogenes). Optionally, the target bacteria is Campylobacter (e.g. Campylobacter jejuni). Optionally, the target bacteria is Yersinia ( Yersinia ) (e.g. Yersinia pestis ( pestis )). Optionally, the target bacteria is Borrelia ( Borelia ) (e.g. Borrelia burgdorferi ( burgdorferi )). Optionally, the target bacteria is Helicobacter (e.g. Helicobacter pylori). Optionally, the target bacteria is Clostridium (e.g. Clostridium difficile or Clostridium botulinum ( botulinum )). Optionally, the target bacteria is Ehrlichia ( Erlichia ) (e.g. Ehrlichia chafee ( chaffeensis )). Optionally, the target bacteria is Salmonella (e.g. Salmonella typhi ( typhi ) or enterica, e.g. Salmonella typhimurium serovar, e.g. DT104). Optionally, the target bacteria is Chlamydia (e.g. Chlamydia pneumoniae ( pneumoniae )). Optionally, the target bacteria is Parachlamydia ( Parachlamydia ) host. Optionally, the target bacteria is Corynebacterium (e.g. Corynebacterium ciphia ( amycolatum )). Optionally, the target bacteria is Klebsiella (e.g. Klebsiella pneumoniae). Optionally, the target bacteria is Enterococcus (e.g. Enterococcus faecalis or Enterococcus faecium ( faecim ), e.g. linezolid resistant). Optionally, the target bacteria is Acinetobacter (e.g. Acinetobacter baumannii, e.g. multi-drug resistant).

[0468] Further examples of target cells are as follows: -

[0469] 1. Optionally, the target bacteria are, for example, Staphylococcus aureus cells that are resistant to an antibiotic selected from the group consisting of methicillin, vancomycin, linezolid, daptomycin, quinupristin, dalbavancin, and teicoplanin.

[0470] 2. Optionally, the target bacteria are, for example, Pseudomonas aeruginosa cells that are resistant to an antibiotic selected from the group consisting of cephalosporins (e.g., ceftazidime), carbapenems (e.g., imipenem or meropenem), fluoroquinolones, aminoglycosides (e.g., gentamicin or tobramycin), and colistin.

[0471] 3. Optionally, the target bacteria are, for example, Klebsiella (e.g., Klebsiella pneumoniae) cells that are resistant to carbapenems.

[0472] 4. Optionally, the target bacteria are, for example, Streptococcus (e.g., Streptococcus thermophilus, Streptococcus pneumoniae, or Streptococcus pyogenes) cells that are resistant to an antibiotic selected from the group consisting of erythromycin, clindamycin, beta-lactams, macrolides, amoxicillin, azithromycin, and penicillin.

[0473] 5. Optionally, the target bacteria are, for example, Salmonella (e.g., typhoid serotype) cells that are resistant to an antibiotic selected from the group consisting of ceftriaxone, azithromycin, and ciprofloxacin.

[0474] 6. Optionally, the target bacteria are, for example, Shigella cells that are resistant to an antibiotic selected from the group consisting of ciprofloxacin and azithromycin.

[0475] 7. Optionally, the target bacteria are, for example, Mycobacterium tuberculosis (M. tb) cells that are resistant to an antibiotic selected from the group consisting of isoniazid (INH), rifampin (RMP), fluoroquinolones, amikacin, kanamycin, and capreomycin, and azithromycin. Mycobacteriumtuberculosis ) cells.

[0476] 8. Optionally, the target bacteria are, for example, Enterococcus cells that are resistant to vancomycin.

[0477] 9. Optionally, the target bacteria are, for example, Enterobacteriaceae cells that are resistant to an antibiotic selected from the group consisting of cephalosporins and carbapenems.

[0478] 10. Optionally, the target bacteria are, for example, Escherichia coli cells that are resistant to an antibiotic selected from the group consisting of trimethoprim, nitrofurantoin, cephalexin, and amoxicillin.

[0479] 11. Optionally, the target bacteria are, for example, Clostridium (e.g., Clostridium difficile) cells that are resistant to an antibiotic selected from the group consisting of fluoroquinolone antibiotics and carbapenems.

[0480] 12. Optionally, the target bacteria are, for example, Neisseria gonorrhoeae cells that are resistant to an antibiotic selected from cefixime (e.g., oral cephalosporin), ceftriaxone (injectable cephalosporin), azithromycin, and tetracycline.

[0481] 13. Optionally, the target bacteria are, for example, Acinetobacter baumannii cells that are resistant to an antibiotic selected from a beta-lactam, meropenem, and carbapenem.

[0482] 14. Optionally, the target bacteria are, for example, Campylobacter (e.g., Campylobacter jejuni) cells that are resistant to an antibiotic selected from ciprofloxacin and azithromycin.

[0483] 15. Optionally, the target cells produce Beta (β)-lactamase (e.g., ESBL-producing E. coli or ESBL-producing Klebsiella).

[0484] 16. Optionally, the target cells are bacterial cells that are resistant to an antibiotic described in any one of Examples 1-14.

[0485] In one example, the target cells are cells of a species selected from Shigella, Escherichia coli, Salmonella, Serratia ( Serratia ), Klebsiella, Yersinia, Pseudomonas, and Enterobacter. Enterobacter

[0486] Optionally, the composition comprises a carrier cell that is each or in combination capable of conjugative transfer of the first DNA into target cells of two or more species selected from Shigella, Escherichia coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas, and Enterobacter.

[0487] In one example, the growth or proliferation of the carrier cell is reduced by at least 50, 60, 70, 80, 90, or 95%. Optionally, the composition or carrier cell is administered simultaneously or sequentially with an antibiotic that is toxic to the target cells. For example, the antibiotic can be any of the antibiotics disclosed herein.

[0488] Optionally, expression of the NSI is under the control of an inducible promoter that is operable in the target cells. Optionally, expression of the NSI is under the control of a constitutive promoter that is operable in the target cells.

[0489] In embodiments, the first DNA (e.g., comprised by a plasmid) contains a screenable or selectable marker gene. For example, the selectable marker gene is an antibiotic resistance gene.

[0490] ​The carrier bacteria can be bacteria selected from the species or genera appearing in Table 5. For example, the species is found in warm-blooded animals, such as livestock vertebrates. For example, the species is found in humans. For example, the species is found in plants. Preferably, non-pathogenic bacteria that colonize non-sterile parts of the human or animal body, such as the skin, digestive tract, urogenital region, mouth, nasal passages, throat and upper respiratory tract, ear and eye, are used as carrier cells, and in one example, the methods of the application are used against a target cell bacterial infection of such parts of the human or animal body. In another embodiment, the infection is a systemic infection. Examples of particularly preferred carrier bacterial species include, but are not limited to: non-pathogenic strains of E. coli (E. coli F18, S17 and E. coli strain Nissle), various Lactobacillus species (e.g., Lactobacillus casei ( L. casei ), Lactobacillus plantarum ( L. plantarum ), Paracasei lactis ( L. paracasei ), Lactobacillus acidophilus ( L. acidophilus ), Lactobacillus fermentum ( L. fermentum ), Lactobacillus zeae ( L. zeae ) and Lactobacillus gasseri ( L. gasseri )), or other non-pathogenic or probiotic skin or GI tract colonizing bacteria such as Streptococcus ( Lactococcus ), Bifidobacterium ( Bifidobacteria ), Eubacterium ( Eubacteria ) and bacterial minicells, which are nucleoid cells destined to die but still able to transfer plasmids (see; e.g., Adler et al., Proc. Natl. Acad. Sci. USA 57; 321-326, 1970; Frazer and Curtiss III, Current Topics in Microbiology and Immunology 69: 1-84, 1975; U.S. Patent No. 4,968,619 to Curtiss III). In some embodiments, the target recipient cells are pathogenic bacteria comprised by a human, animal or plant, for example on the skin or in the digestive tract, urogenital region, mouth, nasal passages, throat and upper respiratory tract, eye and ear. Of particular interest for targeting and eradication are pathogenic strains of Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus ( Staphylococcus pneumoniae ) and other species, Enterobacter species, Enterococcus species and Mycobacterium tuberculosis. In one example, the target cell genus or species is any genus or species listed in Table 5.

[0491] The present application can be used in a wide variety of settings or environments, such as in therapeutic, agricultural, or other settings, including but not limited to those described in U.S. Patents 6,271,359, 6,261,842, 6,221,582, 6,153,381, 6,106,854, and 5,627,275. Others are discussed herein as well, and still others will be apparent to those skilled in the art.

[0492] Numerous types of plasmids comprising first DNA are suitable for use in the present application. With this in mind, one skilled in the art will appreciate that a single vector bacterial strain can contain more than one type of such plasmid (e.g., differing in the antibacterial agent they encode). Further, in another example, two or more different vector bacterial strains, each containing one or more such plasmids, can be combined for a multi-target effect, i.e., for killing two or more different target species or strains, or for killing cells of the same species or strain of target cells.

[0493] The present application can be used in the treatment of humans as well as in various veterinary, agricultural, horticultural, and food processing applications. For human and veterinary uses, and depending on the cell population or tissue targeted for protection, the following modes of administration of the vector bacteria of the present application are contemplated: topical, oral, nasal, ocular, aural, pulmonary (e.g., via an inhaler), ophthalmic, rectal, urogenital, subcutaneous, intraperitoneal, and intravenous. The bacteria can be supplied as a pharmaceutical composition in a delivery vehicle appropriate to the mode of administration selected for the patient to be treated. As used herein, the term "patient" or "subject" refers to a human or an animal (e.g., an animal that can be used specifically as a model for clinical efficacy of a particular donor strain, or is a farm or livestock animal). Commercially relevant animals are chickens, turkeys, ducks, catfish, salmon, cod, herring, lobster, shrimp, prawn, cattle, sheep, goats, swine, goats, geese, or rabbits.

[0494] For example, to deliver the carrier bacteria to the gastrointestinal tract or to the nasal passages, a preferred mode of administration can be by oral ingestion or nasal aerosol, or by feeding (alone or incorporated into the subject's feed or food and / or beverage such as drinking water). In this regard, the carrier cells can be included in the food of the livestock (or domesticated animal or companion animal), e.g., the carrier bacteria are included in a feed additive for the livestock. Alternatively, the additive is a beverage (e.g., water) additive for the livestock. It should be noted that probiotic bacteria such as Lactobacillus acidophilus are sold as gel capsules containing a lyophilized mixture of bacterial cells and a solid support such as mannitol. When the gel capsule is ingested with a liquid, the lyophilized cells rehydrate and become viable clonogenic bacteria. Thus, in a similar manner, the carrier bacteria cells of the present application can be provided as a powdered, lyophilized preparation in a gel capsule or bulk, e.g., for sprinkling onto food or beverage. The rehydrated, viable bacterial cells then colonize and / or colonize sites throughout the upper and / or lower gastrointestinal system, and thereafter contact the target bacteria.

[0495] For topical application, the carrier bacteria can be formulated as an ointment or cream for application to the affected skin surface. Ointment or cream formulations are also suitable for rectal or vaginal delivery, along with other standard formulations such as suppositories. Suitable formulations for topical, vaginal or rectal administration are well known to pharmaceutical chemists.

[0496] The present application can be particularly useful for topical or mucosal administration to treat various bacterial infections or bacterial-related undesirable conditions. Some representative examples of these uses include treatment of: (1) conjunctivitis caused by Haemophilus species, and corneal ulcers caused by Pseudomonas aeruginosa; (2) otitis externa caused by P. aeruginosa; (3) chronic sinusitis caused by a number of gram-positive cocci and gram-negative bacilli, or for general decontamination of the bronchial passages; (4) cystic fibrosis associated with P. aeruginosa; (5) enteritis caused by H. pylori (e.g., to treat or prevent gastric ulcers), E. coli, Salmonella typhimurium, Campylobacter species, or Shigella species; (6) open wounds, e.g., surgical or non-surgical, e.g., as a prophylactic measure; (7) burns, to eliminate P. aeruginosa or other gram-negative pathogens; (8) acne, e.g., caused by P. acnes (9) nasal or skin infections, e.g., caused by methicillin-resistant Staphylococcus aureus (MSRA); (10) body odor, e.g., caused by gram-positive anaerobes (i.e., use of the carrier cells in deodorants); (11) bacterial vaginosis, e.g., associated with Gardnerella vaginalis (12) gingivitis and / or caries, caused by various organisms. Propionobacteracnes Gardnerella vaginalis

[0497] ​​In one example, the target cell is an E. coli cell and the disease or condition to be treated in a human is a urinary tract infection or a ventilator-associated infection, such as pneumonia.

[0498] In other embodiments, the vector cells of the application can be used to treat a surface for removal or attenuation of an unwanted target bacteria, for example, in a method for treating a surface or environment comprising the target bacteria, wherein the method comprises contacting the surface or environment with the vector bacteria of the application, allowing the first DNA of the application to be transferred from the vector to the target bacteria by conjugation, and allowing the antibacterial agent to kill the target cells. For example, a surface that can be used in an invasive procedure, such as surgery, catheterization, etc., can be treated to prevent the subject from becoming infected with bacterial contaminants on the surface. It is contemplated that the methods and compositions of the application can be used to treat a wide variety of surfaces, objects, materials, etc. (e.g., medical or emergency equipment, day care and kitchen equipment and surfaces) to control bacterial contamination thereon.

[0499] Pharmaceutical preparations or other compositions comprising the vector bacteria can be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit of pharmaceutical preparation suitable for use in treating a patient or plant or environment or surface. Each dosage should contain a quantity of vector bacteria calculated to produce the desired antibacterial effect associated with the selected vector. Procedures for determining the appropriate dosage unit are well known to those of skill in the art. Dosage units can be scaled up or down based on the weight of the patient, plant, surface or environment. Appropriate concentrations for achieving eradication of pathogenic target cells (e.g., contained by the tissues of a patient) can be determined by dose concentration curve calculations, as known in the art.

[0500] Other uses of the vector bacteria of the application are also contemplated. These include various agricultural, horticultural, environmental and food processing applications. For example, in agriculture and horticulture, various plant pathogens can be targeted in order to minimize plant disease. One example of a plant pathogen suitable for targeting is Erwinia (e.g., E. amylovora, the causative agent of fire blight). E amylovora Similar strategies can be used to reduce or prevent wilting of cut flowers. For veterinary or animal husbandry, the vector cells of the application can be incorporated into animal feed (chicken, pig, poultry, goat, sheep, fish, shellfish or cattle feed) to reduce the bioburden or eliminate certain pathogenic organisms (e.g., Salmonella in chickens, turkeys or other poultry). In other embodiments, the application can be applied to meat or other food to eliminate unwanted bacteria or pathogens (e.g., E. coli 0157:H7 on meat, or Proteus species, a cause of seafood "fishiness"). Proteus

[0501] ​Environmental utility includes, for example, engineering the carrier bacteria, such as Bacillus thuringiensis and one of its conjugative plasmids, to deliver and conditionally express insecticides in addition to or instead of antimicrobials (e.g., for control of mosquitoes that spread malaria or the West Nile virus). In such applications, as well as in agricultural and horticultural or the other applications described above, it is contemplated that the carrier bacteria be formulated as a solution, aerosol or gel capsule.

[0502] In preferred embodiments of the application, certain features are employed in the DNA, plasmids and carrier cells of the application to minimize potential risks associated with using engineered DNA or genetically modified organisms in the environment. For example, it can be advantageous to utilize DNA or plasmids that are not self-transmissible in environmentally sensitive situations. Instead, the DNA or plasmid can be mobilized by conjugative mechanisms, but not self-transmissible. In some embodiments, this can be accomplished by integrating all or some of the tra genes, the products of which are necessary for assembly of the conjugative machinery, into the carrier cell chromosome. In such embodiments, the DNA or plasmid of the application is configured to have an origin of transfer (oriT), but not the tra genes provided on the chromosome of the cell. This feature prevents the recipient killer cell from further transferring the killer DNA or plasmid before or even after it dies. Another biosafety feature includes utilizing conjugative systems that have a predetermined host range. Certain elements are known to function in only a few related bacteria (narrow host range), while others are known to function in many unrelated bacteria (broad host range or promiscuous) (del Solar et al., MoI. Microbiol. 32: 661-666, 1996; Zatyka and Thomas, FEMS Microbiol. Rev. 21: 291-319, 1998). In addition, many of these conjugative systems can function in Gram-positive or Gram-negative bacteria, but generally not in both (del Solar, 1996, supra; Zatyka and Thomas, 1998).

[0503] In embodiments, the inadvertent spread of antibiotic resistance can be minimized by avoiding the use of antibiotic resistance markers on the DNA or plasmid of the invention that are transferred into target cells by conjugation. In an alternative approach, the genes responsible for the synthesis of an amino acid, i.e. serine, can be mutated, resulting in a requirement for this amino acid in the donor. Such mutant bacteria will prosper on media lacking serine, provided they contain a plasmid with a ser gene whose product is required for growth. Thus, the present invention contemplates the advantageous use of plasmids containing a ser gene or another nutritional genetic marker. These markers will allow selection and maintenance of the DNA or plasmid in the carrier cell. Another bio-safety approach involves the use of restriction modification systems to regulate the host range of the DNA or plasmid. Conjugation and plasmid establishment are expected to occur more frequently between taxonomically related species in which the plasmid can escape the restriction system and replicate. Type II restriction endonucleases make double-stranded breaks within or near specific recognition sequences of duplex DNA. Homologous modification enzymes can methylate the same sequence and protect it from cleavage. Restriction modification systems (RMs) are ubiquitous in bacteria and archaea, but absent in eukaryotes. Some RM systems are plasmid-encoded, while others are on the bacterial chromosome (Roberts and Macelis, Nucl. Acids Res. 24: 223-235, 1998). When foreign DNA, such as viral or plasmid DNA, is not modified by the appropriate modification enzyme, restriction enzymes cleave this DNA. In this way, cells are protected from foreign DNA. Thus, by using a carrier strain that produces one or more methylation enzymes, cleavage by one or more restriction enzymes can be avoided. Site-directed mutagenesis is used to produce plasmid DNA that lacks specific restriction sites or contains new sites, respectively, protecting the plasmid DNA from nucleases or making the plasmid DNA susceptible to nucleases. Broad host range plasmids (e.g. RP4) can simply escape the restriction system by not having many restriction cleavage sites that are normally present on narrow host plasmids (Willkins et al., 1996, J. Mol. Biol 258, 447-456).

[0504] The preferred embodiments of the present application also utilize environmentally safe bacteria as carriers. For example, DNA vaccine delivery by attenuated intracellular gram-positive and gram-negative bacteria has been reported (Dietrich et al., 2001 Vaccine 19, 2506-2512; Grillot-Courvalin et al., 1999 Current Opinion in Biotech. 10, 477-481). In addition, the donor strain can be one of the thousands of harmless bacteria that colonize non-sterile parts of the body (e.g., skin, gastrointestinal tract, urogenital tract, mouth, nasal passages, throat, and upper respiratory system). Examples of preferred donor (i.e., carrier) bacterial species are set forth above.

[0505] In another strategy, non-dividing, non-growing carrier cells are used in place of live cells. Minicells and maxicells are well-studied model systems of metabolically active bacterial cells that cannot survive. Minicells lack chromosomal DNA and are generated by specialized mutant cells that undergo cell division without DNA replication. If the cells contain multicopy plasmids, many minicells will contain plasmids. Minicells neither divide nor grow. However, minicells with conjugative plasmids are able to conjugative replicate and transfer plasmid DNA to live recipient cells. (Adler et al., 1970, supra; Frazer and Curtiss, 1975, supra; U.S. Patent No. 4,968,619, supra). Maxicells can be obtained from E. coli strains carrying mutations in key DNA repair pathways (recA, uvrA, and phr). Because maxicells lack so many DNA repair functions, they die upon exposure to low doses of UV. Importantly, plasmid molecules (e.g., pBR322) that are not exposed to UV continue to replicate. Under such conditions, transcription and translation (plasmid-directed) can occur efficiently (Sancar et al., J. Bacteriol. 137: 692-693, 1979), and proteins made prior to irradiation should be sufficient to support conjugation. This is supported by two observations: i) streptomycin-killed cells are still active donors, and ii) transfer of conjugative plasmids can occur in the presence of antibiotics that prevent de novo gene expression (Heineman and Ankenbauer, 1993, J. Bacteriol. 175, 583-588; Cooper and Heineman, 2000. Plasmid 43, 171-175). Accordingly, UV-treated maxicells are able to transfer plasmid DNA to live recipients. It should also be noted that the conservation of recA and uvrA genes in bacteria should allow for the generation of maxicells from donor strains other than E. coli.

[0506] Also contemplated for use in the application are any modified bacteria because the bacteria contain temperature-sensitive mutations in genes encoding essential cellular functions (e.g., cell wall, protein synthesis, RNA synthesis, as described in, e.g., U.S. Patent 4,968,619, supra) so they cannot function.

[0507] In an alternative, archaea are used instead of bacteria for the vector cell. Additionally or alternatively, the target cell is an archaeal cell.

[0508] As used herein, the term "vector cell" includes dividing and / or non-dividing bacterial cells (minicells and maxicells), or conditionally non-functional cells.

[0509] In one example, the first DNA is comprised by a modified RK2 plasmid (i.e., an RK2 plasmid that has been modified by recombinant DNA techniques or a descendant of such modified plasmid). Plasmid RK2 is a promiscuous plasmid that can replicate in 29 (and possibly more) gram-negative species (Guiney and Lanka, 1989, pp. 27-54. In C. M. Thomas (ed.) Promiscous plasmids in gram-negative bacteria. London, Ltd London, UK.). Plasmid RK2 is a 60-kb self-transmissible plasmid with a known complete nucleotide sequence (Pansegrau et al., 1994, J. Mol. Biol. 239, 623-663). A minimal replicon derived from this large plasmid has been obtained, which lacks all of its genes except the trfA gene, which encodes the Rep protein of the plasmid called TrfA, and the vegetative origin of replication, oriV. For a review on RK2 replication and its control by the TrfA protein, see Helinski et al., 1996 (In Escherichia coli and Salmonella Cellular and Molecular Biology, Vol. 2 (Eds. F. Neidhardt et al., 2295-2324, ASM Press, Washington D.C.).

[0510] In one example, the first DNA is comprised by a modified R6K plasmid (i.e., an R6K plasmid that has been modified by recombinant DNA techniques or a descendant of such modified plasmid).

[0511] The application is optionally used in industry or for household use, or in a method for such use. For example, it is or can be used in the agricultural, oil or petroleum industry, food or beverage industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aerospace industry, automotive industry, biotechnology industry, medical industry, health care, dental industry, energy industry, consumer goods industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or papermaking industry, textile industry, clothing industry, leather or suede or animal skin industry, tobacco industry or steel industry.

[0512] The application is optionally used in industry or in an environment that is an industrial environment, wherein the industry is an industry in a field selected from the group consisting of: medical and health care; pharmaceutical; human food; animal food; plant fertilizer; beverage; dairy; meat processing; agriculture; animal husbandry; poultry farming; fish and shellfish farming; veterinary; oil; natural gas; petrochemistry; water treatment; sewage treatment; packaging; electronics and computers; personal health care and hygiene; cosmetics; dentistry; non-medical dentistry; ophthalmology; non-medical ophthalmology; mineral mining and processing; metal mining and processing; quarrying; aviation; automotive; railway; shipping; aerospace; environment; soil treatment; pulp and papermaking; clothing manufacturing; dye; printing; adhesive; air treatment; solvent; biological defense; vitamin supplement; refrigeration; fiber degumming and production; biotechnology; chemistry; industrial cleaning product; household cleaning product; soap and detergent; consumer product; forestry; fishing; leisure; recycling; plastic; skin, leather and suede; waste management; funeral and mortuary; fuel; construction; energy; steel; and tobacco industry.

[0513] In one example, the first DNA comprises a CRISPR array targeting the target bacteria, wherein the array comprises one, or two or more different spacers (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more spacers) for targeting the genome of the target bacteria.

[0514] In one example, the target bacteria are comprised by an environment. In one example, the environment is a human microbiome, for example an oral microbiome or a gut microbiome or blood stream. In one example, the environment is not an environment in or on a human. In one example, the environment is not an environment in or on a non-human animal. In one embodiment, the environment is an air environment. In one embodiment, the environment is an agricultural environment. In one embodiment, the environment is an oil or petroleum recovery environment, for example an oil or petroleum field or well. In one example, the environment is an environment in or on a food or beverage for consumption by a human or non-human animal. In one example, the environment is a marine environment, for example in seawater or on a ship (e.g. in ship or boat ballast water).

[0515] In one example, the environment is a human or animal microbiome (e.g., gut, vaginal, scalp, axillary, skin, or oral microbiome). In one example, the target bacteria are comprised by a human or animal microbiome (e.g., gut, vaginal, scalp, axillary, skin, or oral microbiome).

[0516] In one example, the carrier bacteria or composition of the application is administered intranasally, topically, or orally to a human or non-human animal, or is for such administration. A skilled artisan aiming to treat a microbiome of a human or animal is able to determine the optimal route of administration, depending on the microbiome of interest. For example, when the microbiome is a gut microbiome, the administration can be intranasal or oral. When the microbiome is a scalp or axillary microbiome, the administration can be topical. When the microbiome is in the mouth or throat, the administration can be oral.

[0517] In one example, the environment is comprised by a beverage or water (e.g., 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.

[0518] In one example, the carrier and / or target bacteria are Firmicutes selected from the group consisting of: Firmicutes ) : Anaerotruncus , Anaerobiospirillum ( Acetanaerobacterium ), Acidovorax ( Acetitomaculum ), Allobaculum ( Acetivibrio ), Anaerococcus ( Anaerococcus ), Anaerofineus ( Anaerofilum ), Anaerocurtis ( Anaerosinus ), Anaerostipes ( Anaerostipes ), Anaerovorax , Butyrivibrio ( Butyrivibrio ), Clostridium, Capracoccus , Dehalobacterium ( Dehalobacter ), Dolosicella ( Dialister ), Dorea ( Dorea ), Enterococcus, Eubacterium ( Ethanoligenens ), Faecalibacterium ( Faecalibacterium ), Fusobacterium ( Fusobacterium ), Gracilibacterium ( Gracilibacter ), Guggenheimella ( Guggenheimella ), Hespellia ( Hespellia ), Lachnospira ( Lachnobacterium ), Lachnoclostridium ( Lachnospira ), Lactobacillus, Leuconostoc ( Leuconostoc ), Megamonas ( Megamonas ), Moryella , Mitsumatusella ( Mitsuokella ), Moryella (Oribacterium ), Acetobacter spp. Oxobacter ), Lactobacillus spp. Papillibacter ), Proprionispira genus *Vibrio pseudobutyricum* Pseudobutyrivibrio ), Pseudomonas spp. Pseudoramibacter ), genus Roselle ( Roseburia ), Rumenococcus ( Ruminococcus ), genus *Dystrophus* Sarcina ), genus Kiyono ( Seinonella ), Shuttleworthia Species of Bacillus ( Sporobacter ), Microsporum spp. ( Sporobacterium Streptococcus, rare Micrococcus ( Subdoligranulum ), Syntrophic cocci ( Syntrophococcus ), Bacillus thermophilus ( Thermobacillus ), Turibacter and Weissella spp. ( Weisella ).

[0519] In one instance, the carrier bacteria, composition, use, or method is used to reduce pathogenic infections or to rebalance intestinal or oral biofilms, for example, to treat or prevent obesity or disease in humans or animals; or to treat or prevent GI conditions (such as Crohn's disease, IBD, or colitis). For example, the DNA, carrier bacteria, composition, use, or method is used to knock down Salmonella, Campylobacter, Erwinia, Xanthomonas, Edwardsiella, Pseudomonas, Klebsiella, and Pectinobacter in the intestinal biofilm of humans or animals. Pectobacterium Clostridium difficile or Escherichia coli bacteria.

[0520] In one example, the animal is, for example, a chicken, and the target bacteria are *Salmonella* or *Campylobacter*. In another example, the animal is, for example, a fish (e.g., catfish or salmon) or a shellfish (e.g., shrimp or lobster), and the target bacteria are *Edwards*. In one example, the plant is a potato plant, and the target bacteria are, for example, *Pectinobacter*. In another example, the plant is a cabbage plant, and the target bacteria are, for example, *Xanthomonas* (e.g., *Xanthomonas spp.*). X campestris In one instance, the plant is a cannabis plant, and the target bacteria is, for example, a genus of Pseudomonas (e.g., *Pseudomonas cannabinoids*). P cannabina ) or Pseudomonas tonsillar ( P amygdali Agrobacterium spp. (e.g., Agrobacterium tumefaciens) A tumefaciens) or Xanthomonas (e.g., Xanthomonas campestris). In one example, the plant is a hemp plant, and the target bacteria is, for example, Pseudomonas (e.g., Pseudomonas syringae or Pseudomonas marginalis), Agrobacterium (e.g., Agrobacterium tumefaciens), or Xanthomonas (e.g., Xanthomonas campestris).

[0521] In one example, the disease or condition is a cancer, an inflammatory or autoimmune disease or condition, for example, obesity, diabetes, IBD, a GI tract condition, or an oral condition.

[0522] Optionally, the environment comprises or the target bacteria comprises an intestinal biofilm, a skin biofilm, an oral biofilm, a throat biofilm, a scalp biofilm, an axillary biofilm, a vaginal biofilm, a rectal biofilm, an anal biofilm, an ocular biofilm, a nasal biofilm, a lingual biofilm, a pulmonary biofilm, a hepatic biofilm, a renal biofilm, a genital biofilm, a penile biofilm, a scrotal biofilm, a mammary biofilm, an auricular biofilm, a urethral biofilm, a labial biofilm, an organ biofilm, or a dental biofilm. Optionally, the environment comprises or the target bacteria comprises a plant (e.g., a tobacco, a crop, a fruit plant, a vegetable plant, or a tobacco, for example, on the surface of or contained within the plant), or an environment (e.g., soil or water or a waterway or an aqueous liquid).

[0523] In one example, the vector cell or composition is used to treat a disease or condition in an animal or human, wherein the disease or condition. In one example, the disease or condition is caused by or mediated by infection of a target cell, which is comprised by the subject or patient. In one example, the disease or condition is associated with infection of a target cell, which is comprised by the subject or patient. In one example, a symptom of the disease or condition is infection of a target cell, which is comprised by the subject or patient.

[0524] Optionally, the disease or condition of the human or animal subject is selected from

[0525] (a) a neurodegenerative disease or condition;

[0526] (b) a brain disease or condition;

[0527] (c) a CNS disease or condition;

[0528] (d) memory loss or impairment;

[0529] (e) a heart or cardiovascular disease or condition, for example, a heart attack, stroke, or atrial fibrillation;

[0530] (f) a liver disease or condition;

[0531] (g) a kidney disease or condition, for example, chronic kidney disease (CKD);

[0532] (h) pancreatic disease or condition;

[0533] (i) lung disease or condition, for example cystic fibrosis or COPD;

[0534] (j) gastrointestinal disease or condition;

[0535] (k) throat or mouth disease or condition;

[0536] (l) eye disease or condition;

[0537] (m) genital disease or condition, for example vaginal, labial, penile or scrotal disease or condition;

[0538] (n) sexually transmitted disease or condition, for example gonorrhoea, HIV infection, syphilis or chlamydia infection;

[0539] (o) ear disease or condition;

[0540] (p) skin disease or condition;

[0541] (q) heart disease or condition;

[0542] (r) nose disease or condition

[0543] (s) blood disease or condition, for example anaemia, for example anaemia of chronic disease or cancer;

[0544] (t) viral infection;

[0545] (u) pathogenic bacterial infection;

[0546] (v) cancer;

[0547] (w) autoimmune disease or condition, for example SLE;

[0548] (x) inflammatory disease or condition, for example rheumatoid arthritis, psoriasis, eczema, asthma, ulcerative colitis, colitis, Crohn's disease or IBD;

[0549] (y) autism;

[0550] (z) ADHD;

[0551] (aa) bipolar disorder;

[0552] (bb) ALS [amyotrophic lateral sclerosis];

[0553] (cc) osteoarthritis;

[0554] (dd) congenital or developmental defect or condition;

[0555] (ee) miscarriage;

[0556] (ff) blood clotting conditions;

[0557] (gg) bronchitis;

[0558] (hh) dry or wet AMD;

[0559] (ii) neovascularisation (e.g. tumour or intraocular);

[0560] (jj) common cold;

[0561] (kk) epilepsy;

[0562] (ll) fibrosis, e.g. liver or lung fibrosis;

[0563] (mm) fungal disease or condition, e.g. thrush;

[0564] (nn) metabolic disease or condition, e.g. obesity, anorexia, diabetes, type I or type II diabetes.

[0565] (oo) ulcer, e.g. gastric ulcer or skin ulcer;

[0566] (pp) dry skin;

[0567] (qq) Sjogren's syndrome

[0568] (rr) cytokine storm;

[0569] (ss) deafness, hearing loss or impairment;

[0570] (tt) slow or rapid metabolism (i.e. slower or faster than average for the subject's weight, sex and age);

[0571] (uu) difficulty conceiving, e.g. infertility or sub-fertility;

[0572] (vv) jaundice;

[0573] (ww) skin rash;

[0574] (xx) Kawasaki disease;

[0575] (yy) Lyme disease;

[0576] (zz) allergy, e.g. to nuts, grass, pollen, dust mites, cat or dog fur or dander;

[0577] (aaa) malaria, typhoid, tuberculosis or cholera;

[0578] (bbb) depression;

[0579] (ccc) low intelligence;

[0580] (ddd) microcephaly;

[0581] (eee) malnutrition;

[0582] (fff) conjunctivitis;

[0583] (ggg) pneumonia;

[0584] (hhh) pulmonary embolism;

[0585] (iii) pulmonary hypertension;

[0586] (jjj) bone disorders;

[0587] (kkk) sepsis or septic shock;

[0588] (lll) sinusitis;

[0589] (mmm) stress (e.g. occupational stress);

[0590] (nnn) thalassemia, anemia, von Willebrand's disease or haemophilia;

[0591] (ooo) shingles or cold sores;

[0592] (ppp) menstruation;

[0593] (qqq) low sperm count.

[0594] Neurodegenerative or CNS disease or condition for treatment or prevention by the present application

[0595] In one example, the neurodegenerative or CNS disease or condition is selected from Alzheimer's disease, senile dementia, Down's syndrome, Parkinson's disease, Creutzfeldt-Jakob disease, diabetic neuropathy, Parkinsonism, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy and Creutzfeldt-Jakob disease. For example, the disease is Alzheimer's disease. For example, the disease is Parkinsonism.

[0596] In one example, wherein the methods of the application are practiced 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 facilitating the transmigration of systemic monocyte-derived macrophages and / or Treg cells across the choroid plexus into the brain of the subject, thereby treating, preventing or slowing progression of the disease or condition (e.g. Alzheimer's disease). In one embodiment, the method causes an increase in IFN-g in the CNS system of the subject (e.g. in the brain and / or CSF). In one example, the method restores nerve fibres and / or reduces the progression of nerve fibre damage. In one example, the method restores nerve myelin and / or reduces the progression of nerve myelin damage. In one example, the methods of the application treat or prevent a disease or condition disclosed in WO2015136541, and / or the methods can be used with any of the methods disclosed in WO2015136541 (the disclosure of which is incorporated in its entirety herein by reference, e.g. for the provision of disclosure of such methods, diseases, conditions and potential therapeutic agents which can be administered to a subject for effecting treatment and / or prevention of CNS and neurodegenerative diseases and conditions, e.g. agents such as immune checkpoint inhibitors, e.g. anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed therein).

[0597] Cancer for treatment or prevention by the method

[0598] Cancers that can be treated include tumors that are not vascularized or substantially not vascularized, as well as vascularized tumors. The cancer can comprise a non-solid tumor (e.g. a hematological tumor such as leukemia and lymphoma), or can comprise a solid tumor. Types of cancer to be treated with the application include, but are not limited to, carcinomas, blastomas and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant neoplasms and malignancies such as sarcomas, carcinomas and melanomas. Adult and pediatric tumors / cancers are also included.

[0599] A hematological cancer is a cancer of the blood or bone marrow. Examples of blood (or hematogenous) cancers include leukemias, including acute leukemias (e.g. acute lymphocytic leukemia, acute myeloid leukemia, acute myelocytic leukemia and myeloblasts, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (e.g. chronic myeloid (granulocytic) leukemia, chronic myelocytic leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0600] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors (e.g., sarcomas and carcinomas) include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilm's tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (e.g., glioma (e.g., brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0601] Autoimmune disease for treatment or prevention by the method

[0602] 1. Acute disseminated encephalomyelitis (ADEM)

[0603] 2. Acute necrotizing hemorrhagic leukoencephalitis

[0604] 3. Addison's disease

[0605] 4. Agammaglobulinemia

[0606] 5. Alopecia areata

[0607] 6. Amyloidosis

[0608] 7. Ankylosing spondylitis

[0609] 8. Anti-GBM / Anti-TBM nephritis

[0610] 9. Antiphospholipid syndrome (APS)

[0611] 10. Autoimmune hemolytic anemia

[0612] 11. Autoimmune neutropenia

[0613] 12. Autoimmune dysautonomia

[0614] 13. Autoimmune hepatitis

[0615] 14. Autoimmune hyperlipidemia

[0616] 15. Autoimmune immunodeficiency

[0617] 16. Autoimmune inner ear disease (AIED)

[0618] 17. Autoimmune myocarditis

[0619] 18. Autoimmune oophoritis

[0620] 19. Autoimmune pancreatitis

[0621] 20. Autoimmune retinopathy

[0622] 21. Autoimmune thrombocytopenic purpura (ATP)

[0623] 22. Autoimmune thyroid disease

[0624] 23. Autoimmune urticaria

[0625] 24. Axonal and neuronal neuropathies

[0626] 25. Balo disease

[0627] 26. Behcet’s disease

[0628] 27. Bullous pemphigoid

[0629] 28. Cardiomyopathy

[0630] 29. Castleman disease

[0631] 30. Celiac disease

[0632] 31. Chagas disease

[0633] 32. Chronic fatigue syndrome

[0634] 33. Chronic inflammatory demyelinating polyneuropathy (CIDP)

[0635] 34. Chronic recurrent multifocal osteomyelitis (CRMO)

[0636] 35. Churg-Strauss syndrome

[0637] 36. Cicatricial pemphigoid / benign mucosal pemphigoid

[0638] 37. Crohn’s disease

[0639] 38. Coogan syndrome

[0640] 39. Cold agglutinin disease

[0641] 40. Congenital heart block

[0642] 41. Coxsackie myocarditis

[0643] 42. CREST disease

[0644] 43. Primary mixed cold agglutinin disease

[0645] 44. Demyelinating neuropathy

[0646] 45. Dermatitis herpetiformis

[0647] 46. Dermatomyositis

[0648] 47. Devic's disease (neuromyelitis optica)

[0649] 48. Discoid lupus

[0650] 49. Drusen's syndrome

[0651] 50. Endometriosis

[0652] 51. Eosinophilic esophagitis

[0653] 52. Eosinophilic fasciitis

[0654] 53. Erythema nodosum

[0655] 54. Experimental allergic encephalomyelitis

[0656] 55. Evans' syndrome

[0657] 56. Fibromyalgia

[0658] 57. Fibrosing alveolitis

[0659] 58. Giant cell arteritis (temporal arteritis)

[0660] 59. Giant cell myocarditis

[0661] 60. Glomerulonephritis

[0662] 61. Goodpasture's syndrome

[0663] 62. Granulomatosis with polyangiitis (GPA) (formerly known as Wegener's granulomatosis)

[0664] 63. Graves' disease

[0665] 64. Guillain-Barre syndrome

[0666] 65. Hashimoto's encephalitis

[0667] 66. Hashimoto's thyroiditis

[0668] 67. Hemolytic anemia

[0669] 68. Henoch-Schonlein purpura

[0670] 69. Herpes gestationis

[0671] 70. Hypogammaglobulinemia

[0672] 71. Idiopathic thrombocytopenic purpura (ITP)

[0673] 72. IgA nephropathy

[0674] 73. IgG4-related sclerosing disease

[0675] 74. Immunomodulatory lipoproteins

[0676] 75. Inclusion body myositis

[0677] 76. Interstitial cystitis

[0678] 77. Juvenile arthritis

[0679] 78. Juvenile diabetes (Type 1 diabetes mellitus)

[0680] 79. Juvenile myositis

[0681] 80. Kawasaki syndrome

[0682] 81. Lambert-Eaton syndrome

[0683] 82. Leukocytoclastic vasculitis

[0684] 83. Lichen planus

[0685] 84. Lichen sclerosus

[0686] 85. Ligneous conjunctivitis

[0687] 86. Linear IgA disease (LAD)

[0688] 87. Lupus (SLE)

[0689] 88. Chronic Lyme disease

[0690] 89. Meniere's disease

[0691] 90. Microscopic polyangiitis

[0692] 91. Mixed connective tissue disease (MCTD)

[0693] 92. Mooren's ulcer

[0694] 93. Muir-Torre disease

[0695] 94. Multiple sclerosis

[0696] 95. Myasthenia gravis

[0697] 96. Myositis

[0698] 97. Narcolepsy

[0699] 98. Neuromyelitis optica (Devic's)

[0700] 99. Neutropenia

[0701] 100. Ocular cicatricial pemphigoid

[0702] 101. Optic neuritis

[0703] 102. Palindromic rheumatism

[0704] 103. PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus)

[0705] 104. Paraneoplastic cerebellar degeneration

[0706] 105. Paroxysmal nocturnal hemoglobinuria (PNH)

[0707] 106. Parry Romberg syndrome

[0708] 107. Pearson's syndrome

[0709] 108. Pars planitis (peripheral uveitis)

[0710] 109. Pemphigus

[0711] 110. Peripheral neuropathy

[0712] 111. Perivenous encephalomyelitis

[0713] 112. Pernicious anemia

[0714] 113. POEMS syndrome

[0715] 114. Polyarteritis nodosa

[0716] 115. Autoimmune polyglandular syndromes type I, II and III

[0717] 116. Polymyalgia rheumatica

[0718] 117. Polymyositis

[0719] 118. Postmyocardial infarction syndrome

[0720] 119. Postpericardiotomy syndrome

[0721] 120. Progesterone dermatitis

[0722] 121. Primary biliary cirrhosis

[0723] 122. Primary sclerosing cholangitis

[0724] 123. Psoriasis

[0725] 124. Psoriatic arthritis

[0726] 125. Idiopathic pulmonary fibrosis

[0727] 126. Pyoderma gangrenosum

[0728] 127. Pure red cell anemia

[0729] 128. Raynaud's phenomenon

[0730] 129. Reactive arthritis

[0731] 130. Reflex sympathetic dystrophy

[0732] 131. Reiter's syndrome

[0733] 132. Relapsing polychondritis

[0734] 133. Restless leg syndrome

[0735] 134. Retroperitoneal fibrosis

[0736] 135. Rheumatic fever

[0737] 136. Rheumatoid arthritis

[0738] 137. Sarcoidosis

[0739] 138. Schmidt syndrome

[0740] 139. Scleritis

[0741] 140. Scleroderma

[0742] 141. Sjogren's syndrome

[0743] 142. Sperm and testicular autoimmunity

[0744] 143. Stiff person syndrome

[0745] 144. Subacute bacterial endocarditis (SBE)

[0746] 145. Susac's syndrome

[0747] 146. sympathetic ophthalmia

[0748] 147. Takayasu's arteritis

[0749] 148. temporal arteritis / giant cell arteritis

[0750] 149. thrombocytopenic purpura (TTP)

[0751] 150. Tolosa-Hunt syndrome

[0752] 151. transverse myelitis

[0753] 152. type 1 diabetes mellitus

[0754] 153. ulcerative colitis

[0755] 154. undifferentiated connective tissue disease (UCTD)

[0756] 155. uveitis

[0757] 156. vasculitis

[0758] 157. vesiculobullous dermatosis

[0759] 158. vitiligo

[0760] 159. Wegener's granulomatosis (now known as granulomatosis with polyangiitis (GPA).

[0761] Inflammatory disease for treatment or prevention by the method

[0762] 1. Alzheimer's disease

[0763] 2. ankylosing spondylitis

[0764] 3. arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis)

[0765] 4. asthma

[0766] 5. atherosclerosis

[0767] 6. Crohn's disease

[0768] 7. colitis

[0769] 8. dermatitis

[0770] 9. diverticulitis

[0771] 10. fibromyalgia

[0772] 11. hepatitis​

[0773] 12. Irritable bowel syndrome (IBS)

[0774] 13. Systemic lupus erythematosus (SLE)

[0775] 14. Nephritis

[0776] 15. Parkinson's disease

[0777] 16. Ulcerative colitis.

[0778] Growth promoters and reduced food conversion

[0779] The examples demonstrate that target bacteria can be targeted using an antibacterial agent to promote growth and enhance FCR in poultry. In this example, a guided nuclease system is used to specifically target Salmonella in poultry.

[0780] In a first aspect, there is provided:

[0781] A method of promoting growth in an animal (e.g. a livestock animal, e.g. a poultry animal), the method comprising administering to the animal a guided nuclease system or component thereof, and introducing the system or component into a target bacterium comprised by the animal, wherein the guided nuclease is capable of recognising and modifying (e.g. cleaving) a target nucleotide sequence comprised by the target bacterium, thereby killing or inhibiting growth or proliferation of the target bacterium, and promoting growth in the animal.

[0782] The method is a non-medical method, and the target bacterium present in the animal is capable of inhibiting growth in the animal. Thus, the method reduces the burden of such bacteria in the animal and promotes growth.

[0783] In a second aspect, there is provided:

[0784] A method of enhancing feed conversion ratio (FCR) in an animal (e.g. a livestock animal, e.g. a poultry animal), the method comprising administering to the animal a guided nuclease system or component thereof, and introducing the system or component into a target bacterium comprised by the animal, wherein the guided nuclease is capable of recognising and modifying (e.g. cleaving) a target nucleotide sequence comprised by the target bacterium, thereby killing or inhibiting growth or proliferation of the target bacterium, and enhancing FCR in the animal.

[0785] The method is a non-medical method, and the target bacterium present in the animal is capable of increasing FCR in the animal. Thus, the method reduces the burden of such bacteria in the animal and enhances FCR (i.e. reduces the number of FCR).

[0786] In a third aspect, there is provided:

[0787] A method of promoting growth in an animal (e.g., a livestock animal, e.g., a poultry animal), the method comprising administering to the animal an antibacterial agent that is toxic to Salmonella bacteria, wherein target Salmonella bacteria comprised by the animal are exposed to the agent and are killed, or the growth or proliferation of the target bacteria is inhibited, and growth of the animal is promoted.

[0788] The method is a non-medical method, and the target bacteria present in the animal are capable of inhibiting growth of the animal. Thus, the method reduces the burden of such bacteria in the animal and promotes growth.

[0789] In a fourth aspect: there is provided

[0790] A method of enhancing feed conversion ratio (FCR) in an animal (e.g., a livestock animal, e.g., a poultry animal), the method comprising administering to the animal an antibacterial agent that is toxic to Salmonella bacteria, wherein target Salmonella bacteria comprised by the animal are exposed to the agent and are killed, or the growth or proliferation of the target bacteria is inhibited, and FCR of the animal is enhanced.

[0791] The method is a non-medical method, and the target bacteria present in the animal are capable of increasing FCR of the animal. Thus, the method reduces the burden of such bacteria in the animal and enhances FCR (i.e., reduces the number of FCR).

[0792] In any of these aspects, optionally, the animal is a livestock animal. Optionally, the animal is a bird, e.g., a poultry bird, e.g., a chicken, turkey, goose, or duck. Preferably, the animal is a chicken.

[0793] In any of these aspects, optionally, the bacteria are Enterobacteriaceae bacteria, e.g., Salmonella. For example, the bacteria are Salmonella enterica, Salmonella typhimurium, or Salmonella enteritidis. For example, the Salmonella is any Salmonella species or strain disclosed herein.

[0794] For example, the system, component, or agent is supplied to the animal in the animal’s feed and / or drink (e.g., mixed in the drinking water). When supplied in the drink, the system, component, or agent can be comprised by a carrier bacterium, wherein the carrier bacterium is comprised in the drink in an amount of 1 x 10 3 to 1 x 10 10 (e.g., 1 x 10 4 to 1 x 10 10 ; 1 x 10 4 to 1 x 10 9 ; 1 x 10 4 to 1 x 10 8 ; 1 x 10 4 to 1 x 10 7 ; 1 x 10 3 to 1 x 1010 ; 1 x 10 3 to 1 x 10 9 ; 1 x 10 3 to 1 x 10 8 ; 1 x 10 3 to 1 x 10 7 ; 1 x 10 5 to 1 x 10 10 ; 1 x 10 5 to 1 x 10 9 ; 1 x 10 5 to 1 x 10 8 ; 1 x 10 5 to 1 x 10 7 ; 1 x 10 6 to 1 x 10 10 ; 1 x 10 6 to 1 x 10 9 ; 1 x 10 6 to 1 x 10 8 ; or 1 x 10 6 to 1 x 10 7 ) cfu / ml. When supplied in a beverage, the system, component, or reagent can be comprised by a carrier bacterium, wherein the carrier bacterium is comprised in the beverage in an amount of at least 1 x 10 8 cfu / ml, for example, wherein the animal is a poultry bird, such as a chicken.

[0795] Optionally, the guide nuclease is any guide nuclease disclosed herein, such as a Cas, TALEN, meganuclease, or zinc finger nuclease. In one example, the component is a crRNA or guide RNA operable in a target cell with a cognate Cas nuclease. The Cas nuclease can be any Cas nuclease disclosed herein. The Cas nuclease can be an endogenous Cas of the target cell, or can be encoded by an exogenous nucleic acid administered to the animal.

[0796] The systems, components, and reagents of the present application can be introduced into a target bacterium by bacterial conjugation (e.g., conjugative transfer from a carrier cell to a target cell) or by a bacteriophage, wherein the bacteriophage transduces a nucleic acid encoding the system, component, or reagent into the target cell.

[0797] Optionally, the target bacterium is comprised by any microbiota disclosed herein found in an animal. Preferably, the microbiota is a gut microbiota (e.g., a gut microbiota of a chicken).

[0798] The livestock animal can be any livestock animal disclosed herein, such as a chicken, pig, cow, sheep, farmed fish (e.g., salmon or catfish), or farmed shellfish (e.g., lobster, prawn, or shrimp).

[0799] It is to be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the application. The principal features of this application can be employed in each of the various embodiments and applications without departing from the scope of the application. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this application and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this application pertains. All publications and patent applications and all U.S. equivalent patents and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in the context of the patent application generally means "one or more" unless otherwise indicated. The use of the term "or" in the claims is used to mean "and / or" unless otherwise indicated. In the application, where ever the term "about" is used, this is intended to indicate that the value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects and / or the study subjects over time and / or methodologies used.

[0800] As used in this specification and claim, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0801] As used herein, the term "or" or the like is used in the inclusive sense, i.e., "and / or", unless otherwise stated. As used herein, the term "or its combination" or like terms means all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or its combination" is intended to encompass each of the following: A, B, C, AB, AC, BC, or ABC, and if order is important, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, it is specifically contemplated that combinations including repeating one or more items or steps of the items are expressly enumerated herein, e.g., BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. Those skilled in the art will appreciate that no limitations are intended to items or items included in combination in any manner based on the context in which the items are presented unless expressly stated otherwise based on context.

[0802] Any portion of the disclosure can be read in combination with any other portion of the disclosure unless otherwise apparent from context.

[0803] In light of the present disclosure, all compositions and / or methods disclosed herein can be made and executed without undue experimentation, using the general knowledge of those skilled in the art. Although compositions and methods of the present application have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods and in the steps or in the order of steps of the method described herein without departing from the concept, spirit and scope of the application. All such Examples

[0804] Example 1: Target gene sequence evaluation and crRNA array design

[0805] List of putative target genes in Salmonella

[0806] By careful analysis and decision making, we assembled the following list of target genes for evaluation by using cleavage of CRISPR / Cas components delivered from the vector cell by conjugative plasmid according to the present application.

[0807] 1) pipA pathogenicity island encoded protein: SPI5

[0808] 2) mViM putative virulence factor

[0809] 3) mViN putative virulence factor

[0810] 4) phoP transcribes genes expressed at low Mg+concentrations (OmpR family); virulence transcriptional regulator protein PHOP

[0811] 5) hilA invasion gene transcriptional activator

[0812] 6) BigA putative surface exposed virulence protein

[0813] 7) sugR ATP-binding protein; pathogenicity island encoded protein: SPI3

[0814] 8) rhuM pathogenicity island encoded protein: SPI3

[0815] 9) pipC invasion gene E protein / pathogenicity island encoded protein: SPI5

[0816] 10) pipB pathogenicity island encoded protein: SPI5

[0817] 11) sicP virulence associated chaperone protein

[0818] 12) sopB invasion gene D protein / pathogenicity island encoded protein: SPI5

[0819] 13) marT putative transcriptional regulator protein / pathogenicity island encoded protein: SPI3

[0820] Target selection

[0821] Initial target selection of Salmonella-specific genes identified 13 putative candidates almost exclusively located on Salmonella pathogenicity islands (listed at the beginning of this example). After careful inspection, the list of targets was reduced to 7 genes, namely pipA , pipB , pipC , hilA , marT , sicP and sopB Importantly, we found that the 7 genes do not show significant sequence homology to other genes from Enterobacteriaceae, thus allowing high specificity in targeting Salmonella cells in vivo and in microbiomes of non-target Enterobacteriaceae containing different (non-Salmonella) species. Using the respective functions available in the molecular biology software, the target sites for S. pyogenes Cas9 defined by the presence of the PAM motif (NGG) were extracted. Between 6 and 14 putative target sites were identified in each of the 7 selected genes.

[0822] crRNA design and synthesis

[0823] To select target sites most likely to result in efficient restriction of dsDNA, the presence of nucleotides at key positions was screened in all potential sequences that have been described in available references to have a positive or negative effect on the efficiency of double-stranded cleavage introduced by Cas9. Three publications report such data, although for sgRNAs:

[0824] (1) Doench et al. 2014, Nature Biotechnology

[0825] (2) Gagnon et al. 2014, PLoS One

[0826] (3) Liu et al. 2016, Scientific Reports

[0827] For each gene one target sequence was chosen to be incorporated into the crRNA array, resulting in three arrays to be tested, which genes meet the majority of parameters described in each of the three publications to influence the efficiency of the restriction. The crRNA array was designed based on sequence data from S. pyogenes (published in Deltcheva et al. 2011, Nature). The following sequences were constructed: SEQ ID NO: 1-3.

[0828] Two of these sequences encoding crRNA were assembled together with sequences encoding the homologous tracrRNA and Cas9.

[0829] Cas9 construct (tracrRNA-Cas9-crRNA)

[0830] The chosen sequences encoding crRNA were combined with different versions of the tracrRNA-Cas9 module, resulting in different plasmid collections.

[0831] For constitutive expression of all components (tracrRNA, Cas9 and crRNA) the tracr-Cas fragment based on plasmid pCas9 (Jiang et al. 2013, Nature Biotechnology; sequence presented as SEQ ID NO: 4) was amplified from S. pyogenes gDNA. We ligated this fragment together with the available crRNA encoding fragments and cloned into the high copy number universal cloning plasmid pJet1.2. This series of constructs was named pFS1.

[0832] To determine if this constitutive CRISPR / Cas construct works equally well in a low copy number context we moved the above system into a low copy number plasmid. Thus, the pFS2 construct is based on a low copy number pSC101 originating from pZS21 MCS (Expressys).

[0833] To control the expression of at least one CRISPR / Cas component by means of an inducible promoter we replaced the constitutive Cas9 promoter with the TetR regulated promoter PLtetO-1. The resulting pFS3 plasmid is also based on a low copy number pSC101 originating from pZS21 MCS. The introduced changes are as follows:

[0834] • The spacing between the RBS and the start ATG has been shortened to 7 bp (TAAATAC)

[0835] • The stop codon has been changed from the less efficient TGA C to a tandem TAATAA T sequence

[0836] Finally, we will generate mobilizable versions of the constitutively expressed plasmids like pFS1 and pFS2 mentioned above. To do so, we will clone the transfer origin (oriT) of plasmid RP4 into an intermediate copy number plasmid (p15A origin) carrying chloramphenicol resistance. This construct should be able to be mobilized and transferred by the chromosomal integration conjugation mechanism as present in RP4 and other mobilizable plasmids.

[0837] Further constructs

[0838] To determine the efficiency of dsDNA restriction by the selected crRNA before final selection and transformation into Salmonella, we aim to clone the target gene (or essential parts thereof) into a compatible plasmid. The selected plasmid is pZA31MCS (Expressys) with a p15A origin compatible with the pFS1 / pFS2 / pFS3 constructs described above. The efficiency can then be tested in E. coli by loss of chloramphenicol resistance (or loss of fluorescence if a yfp derivative is present as reporter). The best performing target sequence can then be used in future more targeted plasmids.

[0839] We prepared the following plasmids:

[0840] pFSmob-C tracrRNA / Cas9 - mob - pZA31MCS

[0841] pFSmobSal7 tracrRNA / Cas9 - Salcr7-3 - mob - pZA31MCS

[0842] pFSmobSal3 tracrRNA / Cas9 - Salcr3.2 - mob - pZA31MCS

[0843] pFSmob-C* control based on Salcr7-3 - mob - pZA31MCS

[0844] Plasmid details

[0845] Plasmid name: pFSmob-C

[0846] Description: p15A origin plasmid based on pZA31 MCS (Expressys) carrying a 4768 bp insert (tracrRNA-Cas9-oriT / mob)

[0847] Plasmid length: 6685 bp

[0848] Sequence verified: Yes; insert sequence verified by primer-walking sequencing

[0849] Additional remarks: No crRNA array present; control plasmid

[0850] Plasmid details

[0851] Plasmid name: pFSmobSal7

[0852] Description: p15A origin plasmid based on pZA31 MCS (Expressys) carrying a 5598 bp insert (tracrRNA-Cas9-crRNA-oriT / mob)

[0853] Plasmid length: 7515 bp

[0854] Sequence verified: Yes; insert sequence verified by primer-walking sequencing

[0855] Plasmid details

[0856] Plasmid name: pFSmobSal3

[0857] Description: p15A origin plasmid based on pZA31 MCS (Expressys) carrying a 5341 bp insert (tracrRNA-Cas9-crRNA-oriT / mob)

[0858] Plasmid length: 7258 bp

[0859] Sequence verified: Yes; insert sequence verified by primer-walking sequencing

[0860] Plasmid details

[0861] Plasmid name: pFSmob-C*

[0862] Description: p15A origin plasmid based on pZA31 MCS (Expressys)

[0863] Plasmid length: 7348 bp

[0864] Sequence verified: Yes; insert sequence verified by primer-walking sequencing

[0865] Example 2: Selective removal of unwanted Salmonella strains by conjugation using a guide nuclease

[0866] Research Objectives

[0867] The goal of this study was to selectively remove unwanted Salmonella strains through conjugation experiments. We mobilized the pFSMobSal7 plasmid from E. coli S17 into Salmonella species. This plasmid contains tracr-Cas9, a crRNA encoding array (Sal7-3), and a transfer origin (oriT).

[0868] SUMMARY

[0869] As a proof of concept, conjugation experiments from E. coli to E. coli were first completed prior to mobilization into Salmonella species. The plasmid pFSMobSal7 (chloramphenicol resistant, CmR) was first transformed into a vector strain that allows for mobilization. In this experiment, the pilus producing strain was E. coli S17 (ATCC® 47055™). The conjugation function was provided by the RP4 plasmid integrated into the chromosome. The recipient cell should be nalidixic acid resistant (25 μg / ml), here we used E. coli JM109 (ATCC® 53323™). The targeted Salmonella was FS26 from our Salmonella species collection. The results show good conjugation efficiency from E. coli S17 to E. coli JM109 and from E. coli S17 to Salmonella enterica (FS26). Killing with pFSMob-3 was effective after conjugation with Salmonella as the recipient.

[0870] INTRODUCTION

[0871] The pFSmobSal7 plasmid was derived from the intermediate copy number pZA31MCS (Expressys). In addition to the tracr-Cas9 and crRNA array fragments, a transfer origin (OriT) sequence was synthesized (called mob2) and cloned into pZA31MCS by assembly methods.

[0872] Prior to initiating the conjugation experiments, the E. coli S17 strain was prepared to be electrocompetent by standard protocols (O'Challahan and Charbit, 1990). The plasmids pFSMobsal7 and pFSMobC* were then transformed into E. coli S17 by electroporation and selected in chloramphenicol (Cm30, 30 μg / mL).

[0873] pFSMobSal7 and pFSMobC* were mobilized from donor E. coli S17 to recipient E. coli JM109, as well as to Salmonella species that are nalidixic acid resistant (NalR) and chloramphenicol sensitive (CmS) by mating on filters, followed by incubation at 37°C. The mixture was then plated in Cm30, Nal25 and Cm30+Nal25. The transconjugants were selected in double antibiotic Cm30+ Nal25 (Phornphisutthimas et al., 2007).

[0874] It should be noted that, prior to the initial conjugation experiment, the killing efficiency of the pFSMob plasmids was first tested by transformation in Salmonella.

[0875] Method

[0876] Materials:

[0877] • Donor strain: E. coli S17 transformed with pFSMobSal7 / pFSMobC*

[0878] • Recipient strain: JM109 / Salmonella enterica FS26

[0879] • LB Broth (LB) (Product number L3522, Sigma-Aldrich), LB Cm30 pg / ml

[0880] • LB Agar plates 1%

[0881] • LB Agar plates 1% Cm30 pg / ml

[0882] • LB Agar plates 1% NaL25 pg / ml

[0883] • LB Agar plates 1% Cm30+ NaL25

[0884] • Whatman ® Membrane filters, nylon, pore size 0.45 to 1 pm, diameter 25 mm. Reference number 28420770, supplied by Sigma-Aldrich

[0885] Experiment 1: Mobilization of pFSMobC* from E. coli S17 to E. coli JM109

[0886] As a proof of concept, the conjugation experiment was first done from E. coli S17 containing the control plasmid (pFSMobC*) to E. coli JM109. This experiment followed the protocol from Phornphisutthimas (Phornphisutthimas et al., 2007):

[0887] 1. Grow overnight cultures of donor pFSMobC* (S17) from our collection in LB+Cm30 and grow recipient strain JM109 in LB broth.

[0888] 2. In the morning, prior to starting the experiment, pre-heat LB agar (1%) plates for at least 30 minutes

[0889] 3. Mix donor strain with recipient in a 1 : 1 ratio as follows:

[0890] 4. 50 μl pFSMobC* (S17) + 50 μl JM109

[0891] 5. 50 μl pFSMobC* (S17) + 50 μl LB

[0892] 6. 50 μl LB + 50 μl JM109

[0893] 7. For each reaction, place filter (0.45 μM to 1 μM) on top of pre-heated LB agar plate (1%)

[0894] 8. Load each mating sample onto the filter

[0895] 9. Without shaking, let the plate stand at 37°C for 3 hours, LB is absorbed and bacteria should remain on the filter

[0896] 10. After incubation, place each filter into a universal tube with 1 ml LB broth and vortex well

[0897] 11. Plate 100 μl (and serial dilutions) on Cm30, Nal25 and Cm30+Nal25 and let stand at 37°C overnight.

[0898] Plates on Cm30 give the number of donors. Plates on Nal25 give the number of recipients, while plates on Nal25+Cm30 give the number of transconjugants (i.e. plasmids mobilized from donors to recipients). Plates with only donors and recipients (no mating) are negative controls.

[0899] Results

[0900] 1) After 18 hours at 37°C, when colonies are countable, colonies are counted individually at different dilutions and the number of colonies is expressed as cfu / mL. As expected, no colonies were recovered on the negative control plates (data not shown). The results (Table 1) show a good efficiency of conjugation from E. coli S17 to E. coli JM109.

[0901]

[0902] Table 1. Conjugation of pFSMobC* from E. coli S17 to E. coli JM109. Results were obtained from the mating on filters after 3 hours of incubation at 37°C. a: All controls have been checked. b: Conjugation efficiency is the number of transconjugants / donor cells

[0903] 2) Mobilization of pFSmobC* plasmid in recipient strain was confirmed by PCR. Primers spCas9-6 forward (5'-ATTGTTTGTGGAGCAGCATAAGC) (SEQ ID NO: 8) and mob2 reverse (5'-GCCTCTAGCACGCGTACCATGGGAT) (SEQ ID NO: 9) were used with an annealing temperature of 50°C. Two colonies from the plate with transconjugants have been tested by PCR. A positive control (pFSmobC* in E. coli S17) was also included. Figure 1

[0904] Conclusion 1:

[0905] Conjugation experiments from E. coli to E. coli have shown a good mobilization efficiency. Further optimization tests for E. coli conjugation have shown a higher conjugation efficiency when mating was incubated at 37°C for 6 hours at a 1:4 ratio.

[0906] Experiment 2: Conjugation of pFSMobSal7 from E. coli S17 to Salmonella spp.

[0907] This experiment follows the protocol from Phornphisutthimas (Phornphisutthimas et al., 2007) and Carraro et al., 2017 with some modifications:

[0908] 1. Grow overnight cultures of pFSMobSal7 (S17) and control pFSMobC* (S17) from our collection in LB+Cm30 and recipient strain Salmonella enterica FS26 in LB broth

[0909] 2. In the morning, plates of LB agar (1%) are pre-warmed for at least 30 minutes before starting the experiment

[0910] 3. Mix donor strains with recipients at a 1:4 ratio as follows:

[0911] 4. 100 μl pFSMobC* (S17) + 400 μl Salmonella FS26

[0912] ​5. 100 μl pFSMobSal7 (S17) + 400 μl Salmonella FS26

[0913] 6. 100 μl pFSMobC* (S17) + 400 μl LB

[0914] 7. 100 μl pFSMobSal7 (S17) + 400 μl LB

[0915] 8. 100 μl LB + 400 μl Salmonella

[0916] 9. Spin at 2000xg for 3 minutes

[0917] 10. Resuspend in 200 μl LB

[0918] 11. Spin at 2000xg for 3 minutes

[0919] 12. Resuspend in 25 μl LB

[0920] 13. For each reaction, place filter (0.45 μM to 1 μM) on top of a pre-warmed LB agar plate (1%)

[0921] 14. Load each mating sample onto the filter

[0922] 15. Without shaking, let the plate stand at 37°C for 6 hours, LB is absorbed and bacteria should remain on the filter

[0923] 16. After incubation, place each filter into a universal tube with 1 ml LB broth and vortex well

[0924] 17. Plate 100 μl (and serial dilutions) on Cm30, Nal25 and Cm30+Nal25 and let stand at 37°C overnight.

[0925] The plates of Cm30 give the number of donors. The plates of Nal25 give the number of recipients, while the plates of Nal25+Cm30 give the number of transconjugants (i.e. plasmids mobilized from donors to recipients).

[0926] Results

[0927] - After 18 hours at 37°C, colonies were counted individually at dilution -3 and the number of colonies is expressed as cfu / mL (Table 2). As expected, no colonies were recovered on the negative control plates (data not shown).

[0928]

[0929] Table 2: Results of conjugation experiments of pFSMob plasmid from E. coli S17 to Salmonella enterica FS26. Results obtained from filters mated for 6 hours at 37°C.

[0930] - These data show that the plasmid pFSMobSal7, delivered by conjugation to the target strain, is able to eliminate 100% of Salmonella enterica strain (FS26) compared to the plasmid control pFSMobC*. Figure 2 ).

[0931] Conclusions

[0932] The study shows that conjugation is a good method for delivery from E. coli S17 to Salmonella enterica (FS26) and that pFSMobSal7 is still effective in killing when delivered by conjugation to the recipient. Conjugation is able to eliminate (kill) 100% of Salmonella.

[0933] References

[0934] Carraro N, Durand R, Rivard N, Anquetil C, Barrette C, Humbert M, Burrus V. (2017). The Salmonella genomic island 1 (SGI1) reshapes the mating apparatus of IncC conjugative plasmids to promote self-propagation. PLOS Gen. Salmonella Carraro N, Durand R, Rivard N, Anquetil C, Barrette C, Humbert M, Burrus V. (2017). The Salmonella genomic island 1 (SGI1) reshapes the mating apparatus of IncC conjugative plasmids to promote self-propagation. PLOS Gen.

[0935] O'Challahan D, Charbit A. (1990). High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation. Mol. Gen. Genet., 223; 156-8.

[0936] Phornphisutthimas S, Thamchaipenet A and Panijpan B. (2007). Conjugation in Escherichia coli. Biochem and Mol Bio Education Vol. 35, No. 6, 440-445

[0937] Example 3: Pan-serotype conjugative transfer and killing of Salmonella

[0938] Purpose of the study

[0939] The aim of this study was to test the ability of pFSmobSal7 and pFSmobSal3 plasmids to selectively remove enteric subserotypes of Salmonella enterica in vitro via conjugation.

[0940] Overview

[0941] Eighteen strains of *Salmonella enterica* subspecies *Salmonella enterica*, including *Salmonella typhimurium* (4), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), *Salmonella enterica* (1), and *Salmonella enterica* (1), and *Salmonella enterica* (1), were selected from our collection of *Salmonella* species (Table 3). The bacteria were prepared as competent cells and transformed with two forms of pFSmob plasmids (i.e., pFSmobSal7 and pFSmobSal3), two forms of pFSmob control plasmids (i.e., pFSmob-C* and pFSmob-C), and pZA31MCS (Expressys) as a further control. After electroporation and incubation at 37°C for 2 hours in SOC Outgrowth Medium (NEB, B90205, LOT-10019857), different dilutions of the original solution were added with chloramphenicol (Cm 30 Plate the culture medium (30 μg / mL) onto 1% LB agar plates. Incubate the plates at 37°C for 18 hours. Results are interpreted as colony forming units / mL (CFU / mL).

[0942] introduction

[0943] pFSmobSal7 and pFSmobSal3 plasmids carry 7 target genes on their crRNA arrays, respectively. pipA, pipB, pipC, hilA, sicP, mart, sopB ) and 3 target genes ( pipC, hilA, mart The positive control pFSmob-C, derived from the pFSmobSal7 plasmid, was used; specifically, pFSmob-C did not contain a crRNA array. The control pFSmob-C was derived from the intermediate copy number pZA31MCS (Expressys) used as a further control. Chloramphenicol (Cm...) 30, 30 pg / mL) to perform the selection of positive colonies. It was previously shown that pFSmobSal7 removes in vitro the capacity of Salmonella enterica serovars typhimurium, enteritidis, wilsworthia, montevideo, hadar and bongor by transformation. The objective of this study was to evaluate all Salmonella species serovars present in our collection in order to assess the in vitro capacity of both pFSmobSal7 and pFSmobSal3 plasmids to selectively remove unwanted bacteria; for collecting significant results, 18 different serovars were selected from our collection and tested by electroporation (Table 3). Plasmids pFSmob-C, pFSmob-C* and pZA31MCS (Expressys) were used as controls.

[0944] Methods

[0945] Bacterial strains were recovered from frozen stocks kept at -80°C and incubated at 37°C for 24 hours (Edwards and Ewing, 1986). Agar dilution method was used to test the susceptibility to Cm 30 (30 pg / mL) according to EUCAST clinical breakpoints table. Bacteria were made competent using O'Challahan and Charbit protocol (O'Challahan and Charbit, 1990). All selected strains were transformed with 100 ng of pFSmobSal7, pFSmobSal3, pFSmob-C, pFSmob-C* and pZA31MCS (Expressys); a negative control was also included (competent cells without added DNA) (Table 4). After 18 hours of incubation, the number of countable colonies (number of colonies between 30 and 300) was expressed as number of colonies / mL (CFU / mL) (Table 4).

[0946]

[0947] Table 3. Salmonella species collection. Eighteen strains of Salmonella enterica subsp. enterica serovars typhimurium (4), enteritidis (1), wilsworthia (1), montevideo (1), heidelberg (1), hadar (1), bongor (1), brendenii (1), infantis (1), kentucky (1), senftenberg (1), mbandaka (1), anatum (1), agona (1) and dublin (1) were selected from our Salmonella species collection.

[0948] Results

[0949] According to EUCAST breakpoints (EUCAST, 2013), it was found that all selected bacteria were susceptible to Cm 30 . After electroporation and incubation at 37°C, the number of colonies was counted and expressed as CFU / mL. The results are shown in Table 4. 30(30 μg / mL) (Sigma) agar plates (Fisher Bioreagents, BP1425-500, LOT- 171784) and plated at different dilutions (undiluted, 10 -1 , 10 -2 ) and the plates were incubated at 37°C for 18 hours. When bacteria grew on Cm 30 (30 μg / mL) agar plates, they were considered transformed. Countable colonies (30 to 300) were expressed as CFU / ml (Table 4). As expected, no colonies were recovered on the negative control plates. All serovars except one (FS67, Salmonella dublin) were efficiently transformed by pZA31MCS (Expressys). Almost all serovars were efficiently transformed by both control plasmids (pFSmob-C* and pFSmob-C) with colony numbers ranging from 10 2 to 10 6 CFU / mL.

[0950] For pFSmobSal7 and pFSmobSal3, the killing effect occurred in all serovars and was found to be similar. In most serovars, the reduction in colony numbers was relatively pronounced compared to those transformed by the control plasmids. No significant difference was found between pFSmobSal7 and pFSmobSal3 in their killing effect except for one strain (FS38, Salmonella montevideo).

[0951]

[0952] Table 4. Transformation of 18 Salmonella species serovars. 18 Salmonella species serovars were transformed with pZA31MCS (Expressys), pFSmob-C*, pFSmob-C, pFSmobSal7 and pFSmobSal3. Almost all serovars were efficiently transformed by pZA31MCS (Expressys), pFSmob-C* and pFSmob-C except for a few exceptions. No significant difference was observed in transformation with pFSmobSal7 and pFSmobSal3. Strain FS67, Salmonella dublin, was found to be unable to be transformed with all plasmids used in this study (NT*).

[0953] CONCLUSION

[0954] All Salmonella species serotypes used in this experiment were efficiently made competent and transformed with pZA31MCS (Expressys), pFSmob-C and pFSmob-C* used as controls. As previously observed, Salmonella serotype Typhimurium did not always seem to be transformable by pFSmob-C*. More generally, there was no specific difference between the two pFSmob-derived control plasmids used in this study (10 2 up to 10 6 CFU / mL). On the other hand, the killing effect of pFSmobSal7 and pFSmobSal3 was found to be similar (10 1 up to 10 3 ). In conclusion, in this study, it was demonstrated that pFSmobSal7 and pFSmobSal3 could selectively remove different Salmonella species serotypes compared to control plasmids.

[0955] References

[0956] Edwards PR, Ewing WH. (1986). Identification of Enterobateriaceae (4th ed.). Elsevier, New York.

[0957] EUCAST, European Committee on Antimicrobial Susceptibility Testing. (2013). Breakpoint tables for interpretation of MICs and zone diameters. Available at: http: / / www.eucast.org (last accessed August 16, 2013 CLSI. (2013). Performance standards for antimicrobial susceptibility testing; Twenty-Third Informational Supplement. Version 3.1.

[0958] O'Challahan D, Charbit A. (1990). High efficiency transformation of Salmonella typhimurium and Salmonella typhi by electroporation. Mol. Gen. Genet., 223; 156-8.

[0959] Example 4: Feed conversion improvement in chickens. Second 2-week efficacy, 2-week safety, and 6-week productivity of the combination Trial.

[0960] Study objectives

[0961] • To determine the effect of the antibacterial conjugative plasmid (pFSmobSal7) on the health, welfare and productivity of chickens over 6 weeks.

[0962] Method

[0963] Ross 308 birds were kept under controlled biosecurity conditions and given water and standard commercial feed ad libitum.

[0964] The birds were dosed continuously with either:

[0965] 1. no water (60 birds)

[0966] 2. strain S17 containing pFSmob-C* at 10 8 cfu / ml drinking water (30 birds)

[0967] 3. strain S17 containing pFSmob-C* at 10 8 cfu / ml drinking water (30 birds)

[0968] 4. strain S17 containing pFSmobSal7 at 10 8 cfu / ml drinking water (60 birds)

[0969] 5. strain S17 containing pFSmobSal7 at 10 9 cfu / ml drinking water (30 birds)

[0970] 6. strain S17 containing pFSmobSal7 at 10 10 cfu / ml drinking water (30 birds)

[0971] Bacterial strains were recovered from frozen stocks kept at -78°C and grown on LB agar at 37°C for 24 hours. Cultures were prepared daily in LB broth containing antibiotics to prevent plasmid loss, with shaking at 180 rpm for 16 hours at 37C, followed by centrifugation at 4000 x g for 10 minutes. The medium was removed and the pellet was resuspended in PBS, which was then diluted in PBS to give a solution for dosing to the chickens. g

[0972] In parallel, a group of 30 birds was dosed orally on day 1 with 0.5 ml of 10 5 CFU / mL Salmonella enteritidis strain FS26 (Folium). Birds were checked for Salmonella colonization on day 3 of the experiment by cloacal swab using ISO 6759 method (1). On day 5 of the experiment, 3 of these Salmonella colonized birds (sentinels) were marked and added to each of groups 1-6. Birds were weighed weekly and feed consumption and mortality were recorded.

[0973] ​On days 12 and 19 (7 and 14 days after mixing with the breeders), 15 birds from each group were euthanized. The ceca were removed for examination for Salmonella by ISO 6759. The hock joints and pad scuffing were recorded. Samples of 1 g of liver and cecal contents were snap-frozen.

[0974] The behavior and body weight of 30 birds from each of groups 1 and 4 were then monitored weekly until day 42, at which time they were euthanized and examined as above.

[0975] Results were recorded on paper or dictated via telephone or radio in the bio-secure facility and transcribed to Microsoft Excel TM For purposes of data transformation, birds that did not have bacteria detected were assigned a count of 1 bacteria / g. Counts and weights were log-transformed and statistically analyzed using GraphPad Prism TM . Distribution normality of the data was assessed using D'Agostino and Pearson Omnibus Normality Test, and when non-normal, analyzed using Kruskall-Wallis test with Dunn's multiple comparison post-test. Differences in proportions of birds were analyzed using Fisher's Exact Test.

[0976] Results

[0977] By day 3, all breeders were colonized with Salmonella. These birds, which were used to inoculate the infection in the test groups, had counts of >10 5 cfu / g of feces.

[0978] On day 7, 15 birds / group were euthanized and the ceca were counted for Salmonella Figure 3 ). Figure 4 Data presented for birds positive / negative for Salmonella are shown.

[0979] Birds dosed with S17-pFSmobSal7 (lowest dose) and pFSmob-C* had significantly lower numbers of Salmonella in the ceca Figure 3 and were significantly less likely to have detectable Salmonella in the ceca on day 7 after mixing with the breeders.

[0980] No mortality was seen in any group; no morbidity was observed, and the high dose of S17-mobSal7 was well-tolerated. Birds were scored for positive and negative behavior, but no differences were seen between groups.

[0981] Further from each of the 30 birds in Group 1 and Group 4, until 42 days of age. No significant differences in behavior were again observed during this period. There were no significant differences in feed consumption between groups, and the weights measured at the time of death were not significantly different between groups. At post-mortem, Salmonella was not detected in the ceca by direct enumeration; no tarsal joint or pad lesions were observed. The average carcass weight of birds in the S17-pFSmobSal7 group was 205 g greater than the control group, which was a significant increase Figure 5 ). Since this was associated with equivalent feed consumption between groups, the S17-pFSmobSal7 group had a significantly improved feed conversion ratio (FCR).

[0982] References

[0983] ISO. 2007. ISO 6579:2007: Microbiology of food and animal feedingstuffs - Horizontal method for the detection of Salmonella spp. (ISO 6579:2002+Amd 1 :2007). Geneva, Switzerland.

[0984] Example 5: Non-replicative conjugative plasmid with conditional essential gene markers

[0985] To meet regulatory requirements, any plasmid used is preferably devoid of antibiotic selection markers, and will be non-replicative in the vast majority, if not all, cells other than the host cell (i.e., the vector cell). To render the plasmid non-replicative, the replication system of the broad host range plasmid RK2 (a plasmid with a rather low copy number) is utilized. RK2 replication depends on the presence of a replication protein (TrfA encoded by trfA on RK2) and the binding of TrfA to the plasmid's nutritional origin (oriV), and does not utilize host mechanisms for replication initiation. Physical separation of trfA from the plasmid and incorporation of this gene into the chromosome results in a resulting RK2 oriV-carrying plasmid that is dependent on host-encoded functions. This prevents the plasmid from actively replicating in the target strain after delivery via conjugation. In the rare case where an equivalent IncP plasmid providing trfA is present in the target cell, the two plasmids will compete for available TrfA, resulting in the loss of one plasmid. Since our plasmid in this example further does not carry a beneficial antibiotic resistance marker or the like, and lacks a plasmid addiction system, it will be lost from the progeny quickly.

[0986] As a selection marker in the absence of antibiotic resistance, an enzyme encoding a biosynthetic pathway for an aromatic amino acid will be used aroAThis is a conditionally essential gene, which is generated when the host cell is in an environment where aromatic amino acids are unavailable or derived from free radicals. aroA The gene is essential when grown under the intermediate of the catalytic reaction. aroA The movement of genes from the host genome to the plasmid backbone provides this selection marker. Chromosomes aroA The gene is replaced in the host (vector cell) strain. trfA A copy.

[0987] Knockout in Escherichia coli laboratory strain DH10B aroA The chromosome copy was then replaced with the trfA expression cassette to generate a bacterial test strain for use with the test plasmid. The trfA expression cassette to be used has the sequence of SEQ ID NO: 10.

[0988] The final host (vector) strain, derived from a symbiotic E. coli isolate from chickens, will undergo the same strain construction procedure.

[0989] aroA The coding sequence (SEQ ID NO: 11) was amplified with similar promoter and terminator regions and assembled into a plasmid along with modules of Cas9, tracrRNA, crRNA, and oriV of RK2.

[0990] Additionally, alternative modules, such as restriction-resistant genes that inhibit type I restriction enzymes (ocr for T7, klcA for RK2, ardA from the conjugating plasmid / transposon, ardB from the conjugating plasmid), can be tested in this plasmid configuration to improve DNA stability after transfer into target cells.

[0991] Further, or alternatively, modules encoding essential portions of the type IV secretory system present for conjugation are located on the plasmid, as its absence would result in a non-functional transfer system within the cell. To facilitate conjugation through the host cell, the type IV secretory system present on plasmid RK2 must integrate into the host genome. The essential components of this system are encoded within three operons located on two plasmid sites, tra1 and tra2. Tra1 encodes the traKLM and traJXIHGF operons, and tra2 encodes the trbBCEFGHJL gene. During integration, a regulatory region located in tra1 between the traKLM and traJXIHGF operons is modified. This sequence contains the origin of transfer (oriT), which has a binding site for the TraJ protein responsible for initiating DNA transfer. If this binding site is not altered by mutagenesis to prevent TraJ binding, the transfer of chromosomal DNA fragments during conjugation will be initiated, resulting in the transfer of unwanted genetic information.

[0992] The sequences of the RK2 tra1 and tra2 modules to be used are in SEQ ID NOs 13 and 14.

[0993] Example 6: Anti-Salmonella plasmid construction and testing

[0994] SUMMARY

[0995] The I-E type Cas system from E. coli K12 (MG1655) is a RNA-guided DNAse mechanism, known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein system (CRISPR-Cas), which represents an adaptive immune system of prokaryotes that targets invading foreign genetic material for degradation. The E. coli I-E type system is composed of different Cas proteins (casABCDE12 and cas3) and recognizes a broad variety of PAM sequences with different degrees of efficacy. The main PAM sequences are: 5'-AAG, AGG, ATG, GAG-3'. The I-E type Cas system from E. coli K12 was used to selectively remove Salmonella spp. The system was modified as in E. coli MG1655, the main components of the cas protein complex were expressed by two transcriptional units, cas3 and casABCDE12, respectively. The modifications introduced during the plasmid construction process included the exchange of the natural, regulated promoter element of the casABCDE operon with a constitutive J23114 promoter, and the replacement of the cas1 and cas2 genes, including their natural ribosome binding sites, with the cas3 gene. In practice, these changes resulted in a constitutively expressed CRISPR-Cas module, which was subsequently tested for functional specificity in targeting Salmonella spp. by the addition of a crRNA array.

[0996] Two versions of the GuidedBiotic® plasmid were constructed that differed in the origin of replication (oriV) and antibiotic selection marker. The resulting plasmids were named pFS-Sal-08-rm and pFS-Sal-09-rm and were tested in vitro to selectively inhibit the growth of Salmonella enterica subsp. enterica serovar enterica by conjugative DNA transfer. In the conjugation experiments, pFS-EcoCas3-01-rm and pFS-EcoCas3-03-rm were used as control plasmids for Salmonella-specific growth inhibition.

[0997] Plasmids were constructed that each comprised a nucleotide sequence encoding an I-E type E. coli Cas3, as well as the cognate casA, B, C, D, and E; a CRISPR array operable with Cas3 and comprising a first spacer complementary to the sequence of the invB gene of S. enterica, a second spacer complementary to the sequence of the sicPa second spacer of the sequence complementary to the gene, and sseE a third spacer of the sequence complementary to the gene; RP4 oriT; p15A ori; E. coli proA a gene; and E. coli proB a gene. For more details, see Table 7.

[0998] Method

[0999] crRNA array design

[1000] To design the CRISPR spacers, the target genes were selected focusing on Salmonella-specific pathogenicity islands (SPIs), which are major virulence factors of Salmonella. The selection criteria were conservation within S. enterica, and low to very low occurrence and conservation of the respective genes in other bacteria. These analyses were performed using the Basic Local Alignment Search Tool (BLAST). The cutoff for the number of hits in non-Salmonella species was set to 1000. If the search yielded >1000 hits, the level of conservation was considered high. 100 to 1000 was intermediate, 20 to 100 was low, and if <20 hits, very low. Specifically, genes associated with the Type III protein secretion system T3SS of SPI-1 and SPI-2 were explored as targets, taking care to include gene candidates that are conserved in Salmonella species, but excluding those that are conserved in other bacterial species. Initially, the search was focused on secreted effectors and chaperone proteins, avoiding regulators and functional components of the T3SS.

[1001] Three selected highly conserved gene targets sicP 、 invB and sseE are part of the T3SS locus encoded by Salmonella pathogenicity islands (SPIs), which are sicP and invB in the case of (both chaperone proteins) SPI-1, and sseE (secreted effector) SPI-2. They all occur in only one copy in S. enterica serovar typhimurium strain LT2, and this is the case for all S. enterica, although it cannot be excluded that they are duplicated in some strains.

[1002] The candidate target genes (a) and subsequent spacers (b) were searched on Blast to ensure that they are specific for Salmonella and minimize off-targeting. The search was performed using the RefSeq database, which contains approximately 10600 S. enterica genomes:

[1003] a) Blast search parameters for gene selection:

[1004] - Salmonella conservation: RefSeq DB limited to Salmonella enterica (taxid: 28901), megablast, max hits: 20000, standard parameters

[1005] - Occurrence outside of Salmonella: RefSeq DB limited to bacteria (taxid: 2), exclude Salmonella (taxid: 590), Blastn, max hits 1000, standard parameters

[1006] b) Blast search parameters for spacer sequences (including PAM):

[1007] - Salmonella conservation: RefSeq DB limited to Salmonella enterica (taxid: 28901), blastn, max hits: 20000, standard parameters

[1008] - Occurrence outside of Salmonella: RefSeq DB limited to bacteria (taxid: 2), exclude Salmonella enterica (taxid: 28901), Blastn, max hits 1000, standard parameters, but expect value set to 100 to capture sequences with low but still significant homology.

[1009] A number of spacers targeting different Salmonella enterica genes were initially screened in silico, selected, cloned, and individually tested in vitro for their efficacy in targeting Salmonella enterica subsp. enterica serovar Enteritidis strain FS26 (see Overview of Target Genes and Spacer Selection and Construction and In Vitro Testing of Rep-21: Spacer Reporter System). Three spacer sequences with the best performance against Salmonella species active were selected and combined into a short crRNA array.

[1010] Spacer sequences were derived from three highly conserved genes present in the Salmonella pathogenicity island (SPI): invB , sicP and sseE . Briefly, a limited number of potential spacer sequences were selected and ranked according to sequence features related to effective target DNA restriction, conservation within Salmonella isolates, and lack of DNA homology outside of Salmonella enterica, characterized by the presence of a strong consensus 5'-PAM sequence capable of initiating DNA restriction. Three spacer sequences meeting these criteria were ultimately selected and incorporated into a functional crRNA array. The final completed crRNA array was ordered as a synthetic gene sequence inserted into a standard cloning plasmid (GeneArt, Thermo Fisher Scientific).

[1011] The sequences of the selected genes are shown in SEQ ID NOs: 20-22. In each gene, the spacer is highlighted in bold and the respective PAM is highlighted in italics (in invB case, the spacer was selected on the minus DNA strand (antisense), so the PAM appears 3' of the spacer. The PAM sequences used for spacer selection were AAG, ATG, AGG and GAG. A Blast search was performed for the spacers with their respective PAM (Table 10).

[1012] The non-Salmonella hits were further analyzed for their level of conservation (spacers were accepted when the level of conservation in the off-targets was < 75%). Sequence selection also depended on the case of the mismatch localization in the off-target hits, excluding those sequences showing conserved PAM and seed (first 8 pb of the spacer). Another criterion of our selection also included the type of bacteria in which the hit was present, if present in a pathogen, the spacer was still considered as a possible candidate.

[1013] Each spacer was tested individually in vitro for its efficacy to target the enterica subspecies enteritidis serovar FS26 of Salmonella enterica, and then they were combined together into the array present in plasmid pFS-Sal-09-rm.

[1014] Plasmid assembly

[1015] For homology-based DNA assembly, individual DNA modules carry a 5' and / or 3' extension with exact homology to the adjacent module. The extension is either introduced during the PCR amplification step of the module, or incorporated during the design of the synthetic DNA module. A constitutive J23114 promoter was introduced.

[1016] CasA-E : 4464 bp casA-casB-casC-casD-casE module was amplified from E. coli K12 (MG1655) genomic DNA.

[1017] Cas3 : 2709 bp Cas3 module was amplified.

[1018] crRNA : crRNA array module Sal-crRNA 1 (546 bp) carrying a 25 bp homology extension at the 3' end of the Cas3 module. The sequence of Sal-crRNA array 1 is shown in SEQ ID NO: 15, with the -10 region of the promoter in italics, the direct repeat underlined, and the spacer corresponding to the selected invB , sicP and seeE Salmonella target sequences of SEQ ID NOs: 1-3, respectively.

[1019] Validation and in vitro testing by conjugation of pFS-Sal-08-rm and pFS-Sal-09-rm

[1020] After confirming the sequences of both constructs, plasmids were transformed into a vector strain that allows mobilization via conjugation of the plasmids to evaluate the efficacy of the Cas3 system to selectively kill Salmonella in the presence of the Sal-crRNA array 1. In this study, E. coli strain S17-1 ΔTn7 was used. 100 ng of pFS-Sal-08-rm and pFS-Sal-09-rm were used to transform 50 μΐ of electrocompetent cells of E. coli S17-1 ΔTn7 by electroporation and selection was performed on kanamycin and chloramphenicol, respectively. Transformation with both plasmids resulted in a good transformation efficiency of E. coli S17-1 ΔTN7 (109CFU / mL). The control plasmids, pFS-EcoCas3-01-rm and pFS-EcoCas3-03, lacking the crRNA array were also transformed in the S17-1 ΔTn7 vector strain to be used as controls in the FS26 transformation with pFS-Sal-09-rm and pFS-Sal-08-rm. To confirm the mobilization capacity of the new plasmids, S17-1 vector cells were first used to conjugate E. coli JM109 cells (naleidixic acid, Nal, resistant). The results reported in Table 8 show similar conjugation efficiencies between pFS-EcoCas3-01 and pFS-Sal-09-rm and between pFS-EcoCas3-03 and pFS-Sal-08-rm in E. coli JM109. The number of transconjugants (selected on plates with double antibiotics) divided by the number of recipients (selected on plates with Nal) was used to calculate the conjugation efficiency. 5 -10 6 The transformed CFU / mL). Control plasmids, pFS-EcoCas3-01-rm and pFS-EcoCas3-03, lacking the crRNA array were also transformed in the S17-1 ΔTn7 vector strain to be used as controls in the FS26 transformation with pFS-Sal-09-rm and pFS-Sal-08-rm. To confirm the mobilization capacity of the new plasmids, S17-1 vector cells were first used to conjugate E. coli JM109 cells (naleidixic acid, Nal, resistant). The results reported in Table 8 show similar conjugation efficiencies between pFS-EcoCas3-01 and pFS-Sal-09-rm and between pFS-EcoCas3-03 and pFS-Sal-08-rm in E. coli JM109. The number of transconjugants (selected on plates with double antibiotics) divided by the number of recipients (selected on plates with Nal) was used to calculate the conjugation efficiency.

[1021] Subsequently, the E. coli vector strain S17-1 ΔTn7 previously transformed with the plasmids was used to evaluate if there was a significant growth inhibition of S. enterica FS26 by conjugation with pFS-Sal-08-rm compared to the control plasmid pFS-EcoCas3-03-rm and by conjugation with pFS-Sal-09-rm compared to pFS-EcoCas3-01-rm. Conjugation was performed from E. coli S17-1 ΔTn7 into S. enterica FS26 resistant to nalidixic acid (Nal). The results showed a significant reduction in the number of CFU / mL in the S. enterica strain FS26 conjugated with both pFS-Sal-08-rm and pFS-Sal-09-rm compared to the respective control plasmids (Table 9).

[1022] In summary, the growth inhibition of Salmonella by the two plasmids carrying the Sal-crRNA array 1 generated in this study indicates that the modified Cas module is functional and able to express all individual components of the type I-E cascade complex, the Cas3 nuclease and the crRNA array without negatively affecting the growth of the non-target E. coli host strains S17-1 and JM109. We demonstrated the ability of the E. coli I-E type based plasmids to be transferred from the E. coli S17-1 carrier strain to the Salmonella enterica strain FS26 by conjugation. We observed a significant growth inhibition (>99.9%) of Salmonella transconjugants in the selected strains by conjugation of both pFS-Sal-08-rm and pFS-Sal-09-rm compared to the controls used. Thus, these data highlight the potential of these E. coli I-E type cascade based constructs for the removal of unwanted bacteria, e.g. for the control of zoonoses.

[1023] Example 7: Testing stability and efficacy of the Folium E. coli based product in chickens fed a diet containing Salmonella. traJXIHGF-traKLM-trbBCDEFGHIJKL

[1024] In Example 4, we tested GuidedBiotic®plasmids contained in the carrier cell of the E. coli S17 strain. In this Example 7, we instead tested GuidedBiotic®plasmids (GB plasmid pFS-Sal-09-proAB-rm, Example 6 and Table 7) contained in a different strain of E. coli carrier (strain X). The helper plasmid pCon_aroA carries a functional copy of the conjugation machinery of plasmid RP4 Treatment groups: ), which was found to promote the mobilization of oriT containing plasmids from host cells to recipient cells in vitro.

[1025] Purpose of the study

[1026] • Determine the stability of E. coli strain X with GuidedBiotic®in drinking water over 24 hours.

[1027] • Enumerate the intestinal and organ contamination by Salmonella given in the feed.

[1028] • Determine the efficacy of E. coli X strain containing active GB plasmids in chickens challenged with Salmonella in the feed.

[1029] Summary

[1030] • Immediately after dosing into deionized water containing a stabilizer, E. coli strain X was found at the expected level of 5 x log-8 CFU / mL and had decreased to 8 x log-7 CFU / mL after 24 hours.

[1031] • Challenge in feed (10 4 CFU / g feed) 24 hours post-challenge, low levels (~log-2-5 CFU / g) of Salmonella were found in the crop and cecum on days 1, 3, and 7 post-challenge. Lower levels (~log-1 CFU / g) were also found in ileal contents, liver, and spleen 7 days post-challenge.

[1032] • Active Guided Biotic®, E. coli strain X containing the active GB plasmid, reduced Salmonella counts in the crop 7 days post-challenge (P < 0.03) ~log-1 CFU / g.

[1033] Methods

[1034] Ross 308 birds (30) were housed under controlled biosecurity conditions and given water and standard commercial chow ad libitum. Each experimental group was kept in separate pens with a wood shavings bedding. Birds had a light regime of 18 hours light / 6 hours dark. Temperature and humidity were kept between standard levels shown in Table 11.

[1035] Animal sample collection and plating

[1036] • Low control - no addition to water, 10 4 CFU Salmonella / gram feed (15 birds) for 24 hours

[1037] • GB-Sal - E. coli strain X Guided Biotic® containing the active GB plasmid (pFS-Sal-09-proAB-rm) at 10 8 CFU / mL in the drinking water from days 1-14, 10 4 CFU Salmonella / gram feed (15 birds) for 24 hours

[1038] Guided Biotic® strains were recovered daily from frozen stocks stored at -78°C and grown on LB agar plus chloramphenicol (30 pg / mL) at 37°C for 24 hours. Strain X with the active GB plasmid strain was prepared in Terrific Broth and grown at 37°C with shaking at 180 rpm for 16 hours. OD 600 After measuring the cell number, the cells were centrifuged and resuspended in deionized water containing a stabilizer to obtain a solution for dosing to the chickens (10 8CFU / mL). At the day of sampling, Vac Pac containing blue dye was included in the last 3 hours before collection to confirm water and GB intake. At days 4-14 of the trial, supplemented water samples were collected at the time of GB addition and 24 hours later for counting of GB. Water samples were decimal diluted in PBS and then plated out on MacConkey agar 3 (Oxoid CM0115) and then incubated at 37°C for 24 hours.

[1039] Salmonella strains (FS26, with naliadic acid marker) were recovered from frozen stocks kept at -78°C and grown on LB agar at 37°C for 24 hours. Test cultures were prepared daily in LB broth and grown at 37°C with shaking at 180 rpm for 16 hours. OD 600 After estimation of cell number, cells were centrifuged and resuspended and diluted in phosphate buffered saline to give a solution of 1 x 10 7 CFU / ml was dropped onto the feed at a rate of 1 ml bacterial suspension per 100 g feed while the feed was mixed thoroughly. Four feed samples were collected for counting of Salmonella content.

[1040] Figure 6

[1041] No bird mortality was recorded. Light levels, addition of bedding, temperature, humidity and stocking density were recorded and were not different between treatments. Three birds from each group were euthanized at days 1 and 3 after challenge and 9 birds at day 7 and samples were taken from the crop, ileum, caecum, liver and spleen. Euthanasia, necropsy and dissection times were recorded. Feathers were wetted with water. Birds were dissected with a disposable scalpel which was discarded after dissection and gloves were changed. Samples of 1 g liver and crop, ileum and caecal contents were taken and snap frozen in liquid nitrogen. The scalpel was disposed of after each organ was taken. Samples were homogenized in 9 volumes of phosphate buffered saline, decimal diluted in phosphate buffered saline and checked with agar incubated at 37°C for 16-18 hours. Salmonella was checked in samples which were negative in direct plating by enrichment in 9 volumes of selenite cysteine broth (Oxoid CM0699) at 37°C for 16-18 hours. The enriched broth was then streaked onto XLD agar (CM0469) containing antibiotics as in Table 12 and incubated at 37°C for 16-18 hours.

[1042] Birds in which no bacteria were detected in direct counts were assigned a count of 0 bacteria / g, while those that were negative in direct counts but positive in the enhanced method were assigned 50 CFU / g. Results were recorded to paper or dictated via telephone or radio in the biosecure facility and transcribed to Microsoft Excel. Data were analyzed in GraphPad Prism. Distribution normality of data was assessed using D'Agostino and Pearson's omnibus normality test, and when non-normal, analyzed using the Mann-Whitney U test. Paired data were analyzed using the paired T test.

[1043] Results

[1044] 1. Salmonella in feed

[1045] • The presence of Salmonella in feed (average 9 x log-4 CFU / g) was as expected.

[1046] 2. Guided Biotic® levels in drinking water

[1047] • The GB levels in drinking water analyzed immediately after mixing (average 5 x log-8 CFU / mL) were as planned. Samples taken 24 hours later confirmed an average decrease to 8 x log-7 CFU / mL.

[1048] 3. Effect of Salmonella dose

[1049] • Overall, low levels of Salmonella were found in the crop (~log 1-3 CFU / g) and ceca (~log 4-5 CFU / g) at days 1 and 3 post challenge. At these times, no Salmonella was found in ileum, liver, or spleen samples.

[1050] • At day 7 post challenge, similar levels of Salmonella were again found in the crop and ceca, and lower levels (~log 1) in the ileum, liver, and spleen.

[1051] 4. Effect of active Guided Biotic® (control versus GB)

[1052] • GB reduced (P<0.03) Salmonella counts in the crop 7 days post challenge Table 1: Exemplary bacteria ).

[1053] • There was a weak trend for GB to reduce Salmonella in the crop (P=0.40) and ceca (P=0.50) at day 3, and in the ileum (P=0.58) and ceca (P=0.60) at day 7.

[1054] Akkermansia

[1055] Optionally, a carrier cell is selected from the table and / or a target cell is selected from the table (e.g., where the carrier and target cells have different species; or have the same species, but different strains, or the carrier cell is engineered, but the target cell is wild-type, or vice versa). For example, the carrier cell is an E. coli cell, and the target cell is a C. difficile, E. coli, Eikmenella ( Ruminococcus ), Enterobacteriaceae, Pediococcus ( Faecalibacterium ), Ruminococcus ( Bacteroidetes ), Firmicutes, Bacteroidetes ( Table 7: Function and origin of each genetic element of the pFS-Sal-09-proAB-rm plasmid containing a Type I CRISPR / Cas system. ), Salmonella, Klebsiella, Pseudomonas, Acinetobacter, or Streptococcus cell.

[1056]

[1057]

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[1127]

[1128]

[1129]

[1130]

[1131] Age (days) Under light (°C)

[1132]

[1133] Table 8. Results of conjugation in JM109. Conjugation in E. coli JM109 strain of S17-1::pFS-EcoCas3-01-rm, S17-1::pFS-Sal-09-rm, S17-1::pFS-EcoCas3-03-rm and S17-1::pFS-Sal-08-rm, showing comparable efficiency between the two constructs and the respective control plasmid. The number of transconjugants (CFU / mL) was divided by the number of recipients to calculate the conjugation efficiency.

[1134]

[1135] Table 9. Results of conjugation in FS26. Conjugation in Salmonella enterica strain FS26 of S17-1::pFS-EcoCas3-01-rm, S17-1::pFS-Sal-09-rm, S17-1::pFS-EcoCas3-03-rm and S17-1::pFS-Sal-08-rm was performed in two biological replicates. Results show a significant reduction in Salmonella enterica strain FS26 by conjugation with active constructs when compared to the respective control plasmid. The number of transconjugants (CFU / mL) was divided by the number of recipients to calculate the conjugation efficiency.

[1136]

[1137]

[1138] Table 10. Conservation of selected spacer regions in Salmonella

[1139]

[1140] Table 11

[1141] Ambient temperature in room (°C) Relative humidity (%) Max 1 Over 35 Plating for Salmonella 35 25-30 60-80 1-7 32 22-27 60-80 7-14 29 19-25 40-80 14-21 26 18-25 40-80 21-28 24 18-25 40-80 28-35 - 18-25 40-80 Enrichment for Salmonella - 15-25 40-80

[1142] Table 12. Plating scheme

[1143] XLD + nalidixic acid (25 μg / mL) Selenite broth, plated on XLD + nalidixic acid (25 μg / mL) Direct count - Salmonella Enrichment count - Salmonella ​ ​

Claims

1. Multiple Escherichia coli ( E. coli The use of carrier cells in the preparation of a composition for enhancing the growth or weight of an animal, wherein the method comprises administering to the animal multiple *E. coli* carrier cells, wherein the animal contains bacterial target cells, and each *E. coli* carrier cell contains a first additional DNA encoding an antimicrobial agent that is toxic to the target cells but non-toxic to the *E. coli* carrier cells, the *E. coli* carrier cells being capable of binding and transferring the first additional DNA into the target cells for expression of the antimicrobial agent therein, wherein the first additional DNA is transferred from the *E. coli* carrier cells into the target cells for expression therein to produce an antimicrobial agent that kills or reduces the growth or proliferation of the target cells in the animal, and enhances the growth or weight of the animal. The target cells are Salmonella enterica ( Salmonella enterica ) cells, and The antimicrobial agent (i) comprises a guide nuclease capable of recognizing and modifying a target chromosomal nucleic acid sequence, wherein the nuclease modifies the chromosome to kill the target cell or inhibit the growth or proliferation of the target cell, and (ii) encodes a guide RNA or crRNA for a CRISPR / Cas system, which operates in conjunction with a Cas nuclease in the target cell to cleave the anterior spacer region sequence contained in the target cell. And the prespacer sequence contains genes. invB , sseE and sicP The nucleotide sequence.

2. The use of claim 1, wherein the animal is a livestock animal.

3. The use of claim 1, wherein the target cells comprise *Salmonella enterica* subsp. *enteroides* (…). S enterica subspecies enterica ).

4. The use of claim 1, wherein the method kills multiple different enteric Salmonella enterica subserotypes.

5. The use of claim 4, wherein each serotype is selected from Typhimurium, Enteritidis, Virchow, Montevideo, Heidelberg, Hadar, Binza, Bredeney, Infantis, Kentucky, Seftenberg, Mbandaka, Anatum, Agona, and Dublin.

6. The use of any one of claims 1-5, wherein the *Escherichia coli* (… E coli The carrier cells are F18, Nissle, or S17 Escherichia coli cells.

7. Use according to any one of claims 1-5, wherein the first appendage DNA is contained in a plasmid, wherein the plasmid contains an RP4 transfer origin (oriT) and / or p15A ori.

8. Use according to any one of claims 1-5, wherein the target cells comprise a first strain and a second strain of Salmonella enterica, and each strain comprises a preseptal region sequence, wherein the target cells of the strains are killed.

9. The use of claim 2, wherein the livestock animal is a chicken.

10. The use of claim 9, wherein the chicken is a broiler or a laying hen.

11. Use according to any one of claims 1-5, wherein the DNA is contained in a plasmid, wherein the plasmid is selected from... IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, InclO, Inccl, IncA, IncB, IncC, IncH, IncIa, IncIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT and IncW Plasmid.

12. A composition comprising a plurality of carrier cells for use in a method comprising administering the carrier cells to a subject to treat an infection of pathogenic target cells, wherein each carrier cell is a bacterial cell containing a first appendage DNA encoding an antimicrobial agent, the antimicrobial agent being toxic to the target cells but non-toxic to the carrier cells, the carrier cells being capable of binding and transferring the first appendage DNA into the target cells for expression of the antimicrobial agent therein, wherein the first appendage DNA is transferred from the carrier cells into the target cells for expression therein to produce the antimicrobial agent, thereby killing or reducing the growth or proliferation of target cells in the subject, wherein the target cells are *Salmonella enterica* (Enterozoa). Salmonella enterica ) cells, and the carrier cells are Escherichia coli cells; wherein The antimicrobial agent (i) comprises a guide nuclease capable of recognizing and modifying a target chromosomal nucleic acid sequence, wherein the nuclease modifies the chromosome to kill the target cell or inhibit the growth or proliferation of the target cell, and (ii) encodes a guide RNA or crRNA for a CRISPR / Cas system, which operates in conjunction with a Cas nuclease in the target cell to cleave a pre-spacer region sequence contained in the target cell, and wherein the pre-spacer region sequence contains a gene. invB , sseE and sicP The nucleotide sequence.

13. The composition of claim 12, wherein the composition is administered to the gastrointestinal tract of a subject.

14. The composition of claim 13, wherein the method is performed on a first group of animals, wherein some or all of the animals contain target cells, wherein the spread of target species cells is reduced in the first group; or wherein the spread from the first group of animals to the second group of animals is reduced.

15. The composition of claim 14, wherein the first group of animals is a flock or herd of animals.

16. The composition of any one of claims 12 to 15, wherein the target cells comprise different species of Salmonella that have been killed.

17. The composition of any one of claims 12 to 15, wherein the carrier cell, target cell, or DNA is a carrier cell, target cell, or DNA as defined in any one of claims 1 to 11.

18. The composition of any one of claims 12 to 15, wherein the method is carried out on livestock or chickens.

19. The composition of claim 18, wherein the chicken is a broiler or a laying hen.

20. The composition of any one of claims 12 to 15, wherein the DNA comprises a plasmid, wherein the plasmid is selected from... IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, IncO, Incl, IncA, IncB, IncC, IncH, IncIa, IncIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT and IncW Plasmid.

21. Use of multiple *E. coli* vector cells in the preparation of a composition for killing animal-derived bacterial target cells in an animal, the method comprising administering to the animal multiple vector cells, wherein each vector cell is a bacterial cell containing a first appendage DNA encoding an antimicrobial agent, the antimicrobial agent being toxic to the target cells but non-toxic to the vector cells, the vector cells being capable of binding and transferring the first appendage DNA into the target cells for expression of the antimicrobial agent therein, wherein the first appendage DNA is transferred from the vector cells into the target cells for expression therein to produce the antimicrobial agent, thereby killing or reducing the growth or proliferation of target cells in a subject, wherein the target cells are *Salmonella intestinale* cells and the vector cells are *E. coli* cells, wherein... The antimicrobial agent (i) comprises a guide nuclease capable of recognizing and modifying a target chromosomal nucleic acid sequence, wherein the nuclease modifies the chromosome to kill the target cell or inhibit the growth or proliferation of the target cell, and (ii) encodes a guide RNA or crRNA for a CRISPR / Cas system, which operates in conjunction with a Cas nuclease in the target cell to cleave the anterior spacer region sequence contained in the target cell. And the prespacer sequence contains genes. invB , sseE and sicP The nucleotide sequence.

22. The use of claim 21, wherein the animal is a livestock animal.

23. The use of claim 21, wherein the method reduces Salmonella spp. in the gastrointestinal tract of the subject.

24. The use of claim 21, wherein the method is performed on a first group of animals, wherein some or all of the animals contain target cells, wherein the spread of target species cells is reduced in the first group; or wherein the spread from the first group of animals to the second group of animals is reduced.

25. The composition of claim 24, wherein the first group of animals is a flock or herd of animals.

26. Use according to any one of claims 21 to 25, wherein the target cells comprise different Salmonella species types that have been killed.

27. Use according to any one of claims 21 to 25, wherein the carrier cell, target cell, or DNA is a carrier cell, target cell, or DNA as defined in any one of claims 1 to 11.

28. The use of claim 22, wherein the livestock animal is a chicken.

29. The use of claim 28, wherein the chicken is a broiler or a laying hen.

30. The use of any one of claims 21 to 25, wherein the DNA is contained in a plasmid, wherein the plasmid is selected from... IncFI, IncFII, IncFIll, IncFIV, IncFV, IncM, Inc9, IncO, Incl, IncA, IncB, IncC, IncH, IncIa, IncIc, IncI2, IncIy, IncJ, IncL, IncN, Inc2e, IncO, IncP, IncS, IncT and IncW Plasmid.

31. DNA for use in any one of claims 1 to 11, wherein the DNA is capable of being introduced into a target cell, wherein the DNA encodes an antimicrobial agent that is toxic to the target cell but not to a carrier cell, the carrier cell being capable of binding and transferring a first additional DNA into the target cell for expression of the antimicrobial agent therein, wherein the first additional DNA is transferred from the carrier cell into the target cell for expression therein to produce the antimicrobial agent, thereby killing or reducing the growth or proliferation of the target cell in an animal, wherein the target cell is *Salmonella enterica* (…). Salmonella enterica The cells, and the carrier cells, are Escherichia coli cells. The antimicrobial agent (i) comprises a guide nuclease capable of recognizing and modifying target chromosomal nucleic acid sequences, wherein the nuclease modifies the chromosome to kill the target cell or inhibit the growth or proliferation of the target cell, and (ii) encodes multiple guide RNAs or crRNAs of the CRISPR / Cas system, wherein the guide RNAs or crRNAs are operable in conjunction with Cas nucleases in the target cell to recognize multiple anterior chromosomal spacer sequences contained in the genome of the target cell, wherein the target cell is an intestinal Salmonella cell, and The anterior spacer sequence contains genes. invB, sicP and sseE The nucleotide sequence.

32. The DNA of claim 31, wherein the DNA is contained in a plasmid comprising a transfer origin and a replication origin, the replication origin being operable for DNA replication in a bacterial host cell.

33. The DNA of claim 31 or 32, wherein the DNA comprises SEQ ID NO:

15.

34. The DNA of claim 31 or 32, wherein the DNA comprises a CRISPR repeat sequence and a spacer region sequence, wherein a. Each of the repeating sequences contains SEQ ID NO: 16; and / or b. The spacer sequence comprises one, two, or three sequences selected from SEQ ID NO: 17-19 and their complementary sequences.

35. The DNA of claim 34, wherein the DNA comprises SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 in a 5' to 3' sequence.

36. The DNA of claim 31 or 32, wherein the animal is a chicken.

37. The DNA of claim 36, wherein the chicken is a broiler or a laying hen.

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