Microcapsule comprising a bacteriophage

Abstract

The present invention relates to the field of protection against bacterial infectious diseases by means of compositions comprising bacteriophages. It concerns in particular an antibacterial composition comprising: at least one bacteriophage chosen from the Podoviridae, Myoviridae and Siphoviridae, as an active agent, in a liquid core, a gelled shell encapsulating the liquid core comprising said bacteriophage and comprising dispersed colloidal particles of UV screening agent, in which the UV screening agent is not a soluble antioxidant compound.

Description

Title: [Microcapsule containing a bacteriophage|

[0001] [The present invention relates to the field of protection against bacterial infectious diseases by means of compositions comprising bacteriophages.

[0002] Pathogenic bacteria can infect a variety of hosts: humans, animals, and plants. Antibacterials can treat bacterial infections either by killing the bacteria (bactericidal compounds) or by blocking their multiplication (bacteriostatic compounds). Broadly speaking, antibacterials can be antibiotics of plant or fungal origin, of bacterial origin, or of fossil origin (by extraction or chemical synthesis). Antibacterials can also be of animal or viral origin. In the latter case, they are phages or bacteriophages, which are viruses that infect bacteria.

[0003] Bacteria have the ability to mutate and thus adapt to their environment and antibacterials. For this reason, antibiotic resistance has become a major problem, and awareness of this resistance is leading to the consideration of alternative antibacterial treatments such as bacteriophages. Furthermore, bacteriophages adapt quickly to bacterial mutations without having a negative effect on the commensal flora. Finally, bacteriophages do not infect eukaryotic cells and are therefore harmless to humans, animals, fish, or plants on which they can be used.

[0004] In the field of human health, the administration of bacteriophages since the 1960s (for example, the phage cpX174, a Mycoviridae infecting Escherichia coli) has demonstrated their safety, allowing them to be used more particularly in cases of antibiotic resistance or as a complement to antibiotic therapy. Non-exhaustive examples include ENT infections caused by Pseudomonas aeruginosa such as ear infections, and intestinal infections caused by Escherichia coli.

[0005] In the field of animal nutrition and health, where the trend is towards reducing the use of antibiotics, there is real interest in the use of bacteriophages. For example, in fish farming to combat pathogens of rainbow trout or salmon such as Flavobacterium psychrophilum, or against Yersinia ruckeri which infects salmon. For poultry farms, phage cocktails are used against Salmonella or Clostridium perfringens, such as those developed by Intralytix Inc.

[0006] In the field of plant health, where bacterial infections are responsible for significant economic losses, the majority of existing biocontrol solutions are based on the use of copper, the toxicity and potential side effects of which lead to the search for other biocontrol solutions, including bacteriophages.

[0007] Finally, in the food industry, bacteriophages can be used to control food safety, through their ability to limit the development of pathogens in food processing stages or on surfaces in contact with food. Among other things, Listeria, Shigella, Salmonella, Escherichia coli and Campylobacter jejuni are priority targets for the food industry.

[0008] One of the difficulties when using bacteriophages is their fragility, their sensitivity to the environment. Indeed, they have walls made entirely of proteins, walls that are thinner and more fragile than those of bacteria. In particular, the extreme pH of certain soils or UV radiation, for example from the sun's rays, are factors to be taken into account.

[0009] Improving the stability and maintaining the viability of phages over time is a major challenge for considering their large-scale use. Spray-drying or freeze-drying technologies exist (Matinkhoo et al., 2011, J Pharm Sci; 100(12):5197-205; Jones et al., 2012, Bacteriophage 2: 208-214; Vandenheuvel et al., 2013, Eur J Pharm Biopharm; 84(3):578-82; Leung et al., 2016, Pharmaceutical research, 33(6), 1486-1496; Leung et al., 2017, International journal of pharmaceutics, 521 (1-2), 141-149). Encapsulation technologies designed to improve stability during storage, transport and application were tested.

[0010] In its search for solutions to protect bacteriophages from external attacks such as pH or UV rays during storage or the application, the Applicant experimented with the co-drying of bacteriophages and yeasts, as explained in application FR2110984.

[0011] Surprisingly, the encapsulation of bacteriophages, used as active agents, in a shell comprising colloidal particles of dispersed UV filter, allows the viability of the phages and their antibacterial activity to be preserved. The phages thus encapsulated allow the effective control of bacterial pathogens, advantageously bacterial pathogens directed against humans, animals or plants.

[0012] Thus, the invention relates to an antibacterial composition comprising at least one bacteriophage selected from Podoviridae, Myoviridae and Siphoviridae, as active agent in a liquid core, said liquid core comprising the bacteriophage being encapsulated in a gelled shell comprising dispersed colloidal UV filter particles, in which the UV filter is not a soluble antioxidant compound.

[0013] [Detailed description]

[0014] The terms phages or bacteriophages will be used interchangeably. Phages or bacteriophages are viruses that infect bacteria in a very specific manner and without significant side effects on the host, whether human, animal or plant. Thus, the antibacterial composition that is the subject of the present invention is directed against any potentially pathogenic bacterium, whether the host is a human, an animal or a plant. By antibacterial activity is meant a lytic activity on the part of the bacteriophage on the bacterium following infection of the bacterium by it.

[0015] According to one embodiment, the targeted pathogens are bacteria capable of infecting plants and / or harvested products. Bacterial plant diseases cause very significant losses in agricultural crops and vegetable crops. Few treatments exist, many are copper-based. Iriarte et al. (Bacteriophage, 2012; 2 (4): 215-224) showed that the phage can penetrate the plant and move from the roots to the leaves, persisting in the plant for up to 7 days.

[0016] For diseases of the aerial parts of plants, the targeted pathogens are chosen from: Erwinia spp. or more specifically Erwinia amylovora, Xanthomonas spp., or more specifically Xanthomonas axonopodis var. citri, whose host is citrus and responsible for the disease commonly called Citrus greening, Xylella spp. or more specifically Xylella fastidiosa, capable of infecting around 600 plant species belonging to more than 80 different botanical families: vines, citrus fruits, fruit trees, almond trees, olive trees, cherry trees, coffee trees, avocado trees, alfalfa, oleander trees, oak trees, maple trees, etc., whose disease affecting vines is called Pierce's disease.

[0017] For soil-borne diseases, for which drip or seed-level application is necessary, the targeted pathogens are chosen from Pseudomonas spp. or more specifically Pseudomonas syringae pv. tomato, whose host is the tomato plant and responsible for the disease called bacterial speck, Ralstonia spp. or more specifically Ralstonia solanacearum, whose host is also the tomato plant and responsible for the disease called bacterial wilt.

[0018] For plant seed harvest products, for which application is made directly to the product, the pathogens particularly targeted are Pectobacterium and Dickeya species likely to infect potatoes.

[0019] Erwinia amylovora is a plant pathogenic bacterium responsible for fire blight, a contagious disease affecting apple, pear, and other plants of the Rosacae family (Momol MT; Aldwinckle HS, 2000. Genetic diversity and host range of Erwinia amylovora. In: Vanneste JL, ed. Fire blight the disease and its causative agent, Erwinia amylovora. Wallingford, Oxon UK: GABI Publishing, 55-72; Zwet T van der; Keil HL, 1979. Fire blight, a bacterial disease of Rosaceous plants. Agriculture Handbook, Science and Education Administration USDA. Beltsville, USA:USDA, No. 510:200 pp). A screening of a collection of 33 phages infecting the Erwinia species, for example, allowed the isolation of five phages from the Myoviridae family which are capable of infecting a wide range of bacterial strains of Erwinia amylovora.Said phages, hereinafter called respectively BPH1, 31 Dx, 26C, P2 and 2By, were used for the encapsulation tests of antibacterial compositions according to the invention.

[0020] According to one embodiment, the targeted pathogens are bacteria capable of infecting humans. For example, but not limited to, strains of bacteria selected from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or strains of the genus Salmonella.

[0021] According to one embodiment, the targeted pathogens are bacteria capable of infecting animals, such as Escherichia coli, Salmonella, Campylobacter, Staphylococcus, Flavobacterium psychrophilum, Yersinia ruckeri, Clostridium perfringens. The animals to be treated are cattle as well as sheep, pigs, birds, fish, crustaceans, etc.

[0022] According to one embodiment, the targeted pathogens are pathogens against which the agri-food industry must protect itself, for dairy, meat or fish products: Listeria, Salmonella, Shigella, Escherichia coli, Campylobacter jejuni etc.

[0023] Advantageously, the encapsulation makes it possible to preserve the antibacterial properties of the antibacterial composition according to the invention, as demonstrated by the experimental results reported in the examples below. Indeed, to demonstrate the viability of a phage and to count the phages, it is essential that said phage remains effective against the pathogen against which it is directed.

[0024] The capsules used in the antibacterial composition according to the invention have a shell formed from a liquid solution comprising a biopolymer having gelling properties and dispersed colloidal UV filter particles, in which the UV filter is not a soluble antioxidant compound.

[0025] The microcapsules used in the antibacterial composition according to the invention make it possible to confine the bacteriophages without immobilizing them.

[0026] Microcapsules are well known to those skilled in the art. They can be formed by different techniques and have different shell compositions. Typically, the microcapsules used in the context of the invention are produced according to the manufacturing method described in French patent No. 2939012 and international application WO2010063937 A1.

[0027] They are formed from a liquid core comprising at least one phage constituting the active agent of the antibacterial composition according to the invention. This liquid core may comprise a mixture of different phages.

[0028] The liquid core is encapsulated by a substantially solid gelled envelope called the shell. In particular, said shell is solid. Preferably, the shell of the microcapsules is mainly composed of a biopolymer having gelling properties. In particular, said shell is composed of said biopolymer having gelling properties. This biopolymer in the majority proportion in the shell is the main biopolymer. Such biopolymers having gelling properties are for example alginate, gellan gum, xanthan gum, pectin, chitosan, agar or carrageenan. Preferably, the shell of the microcapsules forming the antibacterial composition according to the invention is mainly composed of alginate, in particular said shell is composed of alginate.

[0029] When in hydrated form, such as for example in suspension in an aqueous solution, the microcapsules of the antibacterial composition according to the invention have an average diameter of between 50 and 4000 μm, preferably between 100 and 2000 μm, more particularly between 200 and 1000 μm, advantageously between 200 and 600 μm. This average diameter can be measured by various techniques well known to those skilled in the art such as particle size analysis based on laser light diffraction, sieving fractionation or optical microscopy imaging. They can also be in dehydrated form. Preferably, the dehydration is partial, and the dehydrated microcapsules preferably have a moisture content of less than 10%, measured by a moisture analyzer after dehydration.

[0030] Preferably, the microcapsules according to the invention are free, that is to say that they are not included in another structure such as a film, a bead, a gel or encapsulated a second time, but that they are in direct contact with the medium which surrounds them, typically a liquid (if they are in suspension for example) or a gas.

[0031] Colloidal UV filter particles are understood to mean particles with an average size between 10 nm and 10 pm, more preferably between 100 nm and 5 pm. In general, the smaller the particles, the more effective they will be against UV. These filters can also help to opacify the microcapsules.

[0032] In the context of the present invention, the UV filter is not a soluble antioxidant compound. Indeed, these compounds tend to degrade more quickly when exposed to UV. The UV filter used in the microcapsules forming the antibacterial composition according to the invention may be organic or inorganic, or a mixture of organic and inorganic filters. Said UV filter is preferably chosen from titanium oxide, carbon black, biochar, charcoal, latex, silica, clays, and mixtures thereof. Biochar is the product of biomass pyrolysis, used as an amendment to agricultural soils. Clays used as UV filters are, for example, talc or kaolin and mixtures thereof. Kaolin is a type of clay also used to protect certain crops from pests and diseases in agriculture.

[0033] Preferably, the UV filter used in the microcapsules forming the antibacterial composition according to the invention is biodegradable and / or edible and / or pharmaceutically acceptable.

[0034] By "dispersed UV filter particles" is meant dispersed particles, preferably in the shell of the microcapsule, in order to allow a homogeneous anti-UV effect on the surface of the microcapsule. Preferably, the core of the capsule according to the invention does not comprise UV filter particles. In particular, the concentration of UV filter is between 0.05 and 30% v / v, more particularly between 0.1 and 20% v / v, relative to the total volume of the shell.

[0035] Preferably, in the methods and uses according to the invention, the microcapsules are used as such, that is to say that they are not included in another structure (such as a film, a bead, a gel or encapsulated a second time), but that they are used directly, possibly in suspension in a liquid.

[0036] The invention also relates to a method for preventing and / or treating plant plants against at least one pathogenic bacterium comprising the steps of: providing an antibacterial composition comprising at least one bacteriophage as an active agent against said at least one pathogenic bacterium, said bacteriophage being protected against UV in a microcapsule comprising colloidal UV filter particles dispersed in the shell, in which the UV filter is not a soluble antioxidant compound, applying said composition to the aerial parts of the plant.

[0037] In particular, said at least one bacteriophage is chosen from Podoviridae, Myoviridae and Siphoviridae.

[0038] According to one embodiment, the harvested products of said plant plants are subjected to prevention and / or antibacterial treatment.

[0039] According to one embodiment, the application of the antibacterial composition can also be carried out by spreading on the soil. According to another embodiment, the application of the antibacterial composition can be carried out around the seeds, in particular but not only by coating or film-coating. According to another embodiment, the application can be carried out on the products after harvest.

[0040] In another embodiment, the invention relates to the use, preferably non-therapeutic, of an antibacterial composition according to the invention for animal and / or human nutrition and / or feed. In this embodiment, the phages used are preferably not therapeutic phages. In other words, said phages are of interest from a nutritional or dietary point of view but do not make it possible to prevent or treat diseases in the subject who consumes them.

[0041] By "food" we mean the habitual or frequent intake of food. Indeed, the antibacterial composition may contain various phages that can improve the properties of a food product, including its bioavailability or shelf life.

[0042] By "nutrition" we mean the taking, often more occasional or as part of a treatment, of a food supplement typically to avoid or compensate for a deficiency. Indeed, the antibacterial composition according to the invention may contain phages varied which allow to improve nutrient intake by improving their bioavailability or even to modify the intestinal flora.

[0043] The invention also relates to a pharmaceutical composition comprising an antibacterial composition according to the invention.

[0044] The present invention also relates to the antibacterial composition or pharmaceutical composition according to the invention for its use as an antibacterial agent, in particular as a medicament, and more particularly for the treatment of a disease caused by a bacterium, in a subject in need thereof. In this embodiment, the antibacterial composition according to the invention advantageously comprises a phage chosen from bacteriophages targeting pathogenic bacteria of humans or animals, and more particularly a bacteriophage targeting bacteria chosen from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or strains of the genus Salmonella.

[0045] The term "pharmaceutical composition" as defined herein means a mixture or solution comprising at least one therapeutic agent to be administered to a subject in order to prevent or treat a particular disease affecting the subject.

[0046] The pharmaceutical compositions as defined herein therefore preferably further comprise pharmaceutically acceptable excipients.

[0047] By "pharmaceutically acceptable" is meant herein compositions and molecular entities that do not produce adverse, allergic or otherwise unwanted reactions when administered to a subject.

[0048] By "subject" we mean here a living being, preferably a mammal, and more particularly a human.

[0049] By "therapeutically effective amount" is meant herein an amount effective, at doses and for periods of time necessary, to achieve the desired therapeutic result. This amount may vary depending on factors such as the disease, the extent of the disease, the age, sex and weight of the subject, and the ability of the microcapsules to cause a desired therapeutic result. A therapeutically effective amount encompasses an amount in which any toxic or deleterious effects are outweighed by the therapeutically effective effects. beneficial. A therapeutically effective amount also includes an amount sufficient to confer a benefit, for example, a clinical benefit.

[0050] Such pharmaceutical compositions are preferably adapted to the route of administration.

[0051] In a particular embodiment, the pharmaceutical compositions are suitable for oral, sublingual, buccal, intranasal or topical administration.

[0052] As mentioned above, the invention also relates to an antibacterial composition or a pharmaceutical composition according to the invention for its use as a medicament. The invention also relates to an antibacterial composition according to the invention for its use for the formulation of pharmaceutical compositions.

[0053] For these uses, the antibacterial composition according to the invention advantageously comprises a phage chosen from bacteriophages targeting pathogenic bacteria of humans or animals, and more particularly a bacteriophage targeting bacteria chosen from Escherichia coll, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or strains of the genus Salmonella.

[0054] Indeed, the use of an antibacterial composition according to the invention makes it possible to facilitate the storage, packaging or preparation of pharmaceutical compositions while preserving the active phages.

[0055] By "treatment" or "treating" is meant herein achieving, partially or substantially, one or more of the following results: partially or totally reducing the extent of the disease, improving a clinical symptom or indicator associated with the disease, delaying, inhibiting or preventing the progression of the disease. In particular, within the scope of the present invention, the disease to be treated is a disease caused by a bacterium, in particular those mentioned above.

[0056] The following examples are provided for illustration purposes only and do not limit the scope of the invention.

[0057] Examples

[0058] Phage counting method The methods for demonstrating the viability and enumerating bacteriophages necessarily use the bacteria targeted by the bacteriophages tested. Generally speaking, the methods for enumerating bacteriophages of unknown concentration are semi-solid agar bilayer methods known to those skilled in the art. A preculture of the target bacteria is carried out in liquid LB medium (Luria Bertani broth). In some cases, the preculture step in liquid medium can be doubled to achieve sufficient volume and concentration, said concentration being assessed by measuring the OD (optical density) at 600 nm. A serial dilution of the phages to be counted is carried out in liquid LB medium. The serial dilutions of phages are brought into contact with the bacterial cultures on LBA medium (Luria Bertani Agar) by first adding semi-solid Luria Bertani Agar medium with 0.75% agar. Reading and counting of lysis plaques are carried out after incubation for 16 to 24 hours at 30°C.

[0059] A Salmonella Newport strain was used for the enumeration of anti-Salmonella phages supplied by Intralytix Inc (Salmofresh™). A Listeria monocytogenes strain was used for the enumeration of anti-Listeria phages supplied by Intralytix Inc. An Erwinia amylovora Ea4 strain provided by INRAe was used for the enumeration of anti-Erwinia phages (phages BPH1, 31 Dx, 26C, P2 and 2By).

[0060] Example 1: Phage encapsulation test and post-encapsulation stability assessment

[0061] The aforementioned BPH1 phage was encapsulated in a capsule without UV filter. Drying tests were performed with three different drying media. Viability and stability over time and temperature were evaluated.

[0062] Among glycerol, SM-sucrose and trehalose tested as drying excipients, SM-sucrose gave the most acceptable drying performance.

[0063] The stability of the encapsulated phage was tested for one month at +4°C, +25°C and +40°C respectively. The titer of the encapsulated phage, in liquid form, decreased very little at +4°C or +25°C. The titer decreased by approximately 2 log at +40°C. In solid form, i.e. after drying with SM-sucrose as an excipient, the titer remained essentially the same regardless of the storage temperature, with a decrease of less than 1 log at +40°C.

[0064] Example 2: Protection of anti-E / w / n / a phages in a capsule including a UV filter

[0065] The above-mentioned phages BPH1, 31 Dx, 26C, P2 and 2By were encapsulated in a shell containing biochar as a UV filter. Phages alone, capsules with or without biochar containing phages were exposed, in liquid form, to UV. The phages were deposited at the CNCM (National Collection of Cultures of Microorganisms, housed at the Pasteur Institute, 25 rue du docteur Roux, 75015 Paris, France) according to the Budapest Treaty: BPH1 filed on December 17, 2021 under number I-5803, 31 Dx filed on May 6, 2022 under number I-5845, 26C filed on May 6, 2022 under number I-5846, P2 filed on May 6, 2022 under number I-5847, and 2By filed on May 6, 2022 under number I-5848.

[0066] The UV exposure protocol was as follows: recovery of a homogeneous sample of capsules with a size of 100 μm using sterile cell sieves. sampling of 3 g of wet capsules or 1 g of dry capsules, respectively, sieved in a sterile Petri dish addition of 0.1 M NaCl buffer diluted 1 / 10 with sterile physiological water, collection of the control before exposure UV exposure with a lamp having undergone the necessary preheating, at an intensity of 105 mW / cm 2 . The Petri dishes are placed in the center of the oven. Exposure times were 10, 20, and 30 minutes for naked phages, encapsulated phages without UV filter, encapsulated phages with UV filter, and the NaCl buffer control, respectively. Two hundred microliters of each UV-exposed sample are taken for counting. Prior to the counting carried out as indicated above, as much supernatant as possible is removed, the capsules are weighed, opened with the addition of 1 mL of PBS EDTA, shaken for 5 minutes on a rotating disk shaker at moderate speed and then serially diluted to proceed with the counting according to the protocol indicated above.

[0067] Table 1 shows the titration results of BPH1 phages after UV exposure times of 0, 5, 10, 20 and 30 minutes respectively. LOD means limit of detection.

[0068] [Table 1] Sample Exposure time (min) CFU / ml negative control 0 LDD free phage 0 4.33E+07 wet capsules without biochar El 0 2.06E+06 wet capsules without biochar E2 0 2.89E+06 wet capsules with biochar El 0 2.89E+06 wet capsules with biochar E2 0 3.67E+06 negative control 5 LDD free phage 5 LDD wet capsules without biochar El 5 LDD wet capsules without biochar E2 5 LDD wet capsules with biochar El 5 4.08E+04 wet capsules with biochar E2 5 3.11E+05 negative control 10 LDD free phage 10 LDD wet capsules without biochar El 10 LDD wet capsules without biochar E2 10 LDD wet capsules with biochar El 10 2.06E+05 wet capsules with biochar E2 10 3.50E+05 negative control 20 LDD free phage 20 LDD wet capsules without biochar El 20 LDD wet capsules without biochar E2 20 LDD wet capsules with biochar El 20 l,28E+05 wet capsules with biochar E2 20 4,28E+04 negative control 30 LDD free phage 30 LDD wet capsules without biochar El 30 LDD wet capsules without biochar E2 30 LDD wet capsules with biochar El 30 3.61E+04 wet capsules with biochar E2 30 4.61E+04,

[0069] These results show that the phage titer decreases drastically (>4log) after 5 minutes of UV exposure, in the same proportion as the “free” phages used as a control. Conversely, the decrease in titer is less significant for phages in capsules with biochar: from 1 log after 5 minutes to 2 log after 30 minutes of exposure). These results clearly show that a capsule containing biochar effectively protects phages against inactivation.

[0070] Encapsulated and dried phages exposed to UV after rehydration achieved similar results after 10 minutes of exposure, as shown in Table 2.

[0071] [Table 2] Sample Exposure time (min) CFU / ml free phage 0 6.00E+07 dry capsules without biochar E2 0 2.51E+06 dry capsules without biochar El 0 3.11E+06 dry capsules with biochar E2 0 l,78E+06 dry capsules with biochar El 0 2.00E+06 free phage 10 l,67E+02 dry capsules without biochar E2 10 l,67E+02 dry capsules without biochar El 10 l,67E+02 dry capsules with biochar E2 10 l,22E+06 dry capsules with biochar El 10 7.33E+05

[0072] Tables 3 and 4 present the titration results of 31 Dx phages after UV exposure times of 0, 5, 10, 20 and 30 minutes, respectively. LOD means limit of detection, for wet capsules and dry capsules, respectively.

[0073] [Table 3]

[0074] [Table 4]

[0075] Tables 5 and 6 present the titration results of 26C phages after UV exposure times of 0, 5, 10, 20 and 30 minutes, respectively. LOD means limit of detection, for wet and dry capsules, respectively.

[0076] [Table 5] Sample Exposure Time Concentration (PFU / MI or PFU / g) Negative Control 0 0.00E+00 Free Phage 0 l.56E+09 Wet Capsules El 0 7.94E+07 Wet Capsules E2 0 8.21E+06 Negative Control 10 0.00E+00 Free Phage 10 2.07E+04 Wet Capsules El 10 3.27E+07 Wet Capsules E2 10 2.89E+06 Negative Control 20 0.00E+00 Free Phage 20 LDD Wet Capsules El 20 l.16E+07 Wet Capsules E2 20 2.22E+06 Negative Control 30 0.00E+00 Free Phage 30 LDD Wet Capsules El 30 l.78E+07 Wet Capsules E2 30 l.72E+06

[0077] [Table 6] Concentration (UFP / MI or sample Exposure time UFP / g) negative control 0 0.00E+00 free phage 0 l.50E+09 dry capsules El 0 1J8E+08 dry capsules E2 0 9.47E+08 negative control 10 0.00E+00 free phage 10 9.07E+04 dry capsules El 10 l.94E+07 dry capsules E2 10 l.13E+08 negative control 20 0.00E+00 free phage 20 LDD dry capsules El 20 4.45E+08 dry capsules E2 20 2.50E+08 negative control 30 0.00E+00 free phage 30 LDD dry capsules El 30 4.68E+07 dry capsules E2 30 4.69E+07

[0078] Tables 7 and 8 present the titration results of P2 phages after UV exposure times of 0, 5, 10, 20 and 30 minutes respectively. LDD means limit of detection, respectively for wet capsules and dry capsules. NA means not applicable (data not available for technical reasons).

[0079] [Table 7] Sample Exposure Time Concentration (PFU / MI or PFU / g) Negative Control 0 0.00E+00 Free Phage 0 l.92E+09 Wet Capsules El 0 l.10E+08 Wet Capsules E2 0 l.24E+08 Negative Control 10 0.00E+00 Free Phage 10 LDD Wet Capsules El 10 9.73E+06 Wet Capsules E2 10 l.04E+07 Negative Control 20 0.00E+00 Free Phage 20 LDD Wet Capsules El 20 3.52E+06 Wet Capsules E2 20 NA Negative Control 30 0.00E+00 Free Phage 30 LDD Wet Capsules El 30 3.82E+06 Wet Capsules E2 30 2.73E+06

[0080] [Table 8] Concentration (IIFP / MI or sample Exposure time UFP / g) negative control 0 0.00E+00 free phage 0 l.69E+09 dry capsules El 0 6.62E+08 dry capsules E2 0 8.04E+07 negative control 10 0.00E+00 free phage 10 5.44E+04 dry capsules El 10 6.06E+08 dried capsules E2 10 8.89E+07 negative control 20 0.00E+00 free phage 20 LDD dry capsules El 20 8.56E+07 dry capsules E2 20 7.20E+08 negative control 30 0.00E+00 free phage 30 LDD dry capsules El 30 1.11E+08 dry capsules E2 30 5.88E+07

[0081] Tables 9 and 10 present the titration results of 31 Dx phages after UV exposure times of 0, 5, 10, 20 and 30 minutes, respectively. LOD means limit of detection, for wet capsules and dry capsules, respectively.

[0082] [Table 9] Sample Exposure Time Concentration (PFU / MI or PFU / g) Negative Control 0 0.00E+00 Free Phage 0 1 2E+07 Wet Capsules El 0 2.41E+06 Wet Capsules E2 0 l.18E+06 Negative Control 10 0.00E+00 Free Phage 10 LDD Wet Capsules El 10 4.09E+06 Wet Capsules E2 10 5.06E+05 Negative Control 20 0.00E+00 Free Phage 20 LDD Wet Capsules El 20 6.57E+04 Wet Capsules E2 20 7.23E+04 Negative Control 30 0.00E+00 Free Phage 30 LDD Wet Capsules El 30 l.71E+05 Wet Capsules E2 30 2.96E+05

[0083] [Table 10] Concentration (UFP / MI or sample Exposure time UFP / g) negative control 0 0.00E+00 free phage 0 5.83E+06 dry capsules El 0 5.14E+06 dry capsules E2 0 4.21E+06 Negative control 10 0.00E+00 free phage 10 LDD dry capsules El 10 1J5E+06 dry capsules E2 10 5.65E+06 Negative control 20 0.00E+00 free phage 20 LDD dry capsules El 20 3.39E+06 dry capsules E2 20 l.58E+06 Negative control 30 0.00E+00 free phage 30 LDD dry capsules El 30 l.22E+06 dry capsules E2 30 6.44E+06 Example 3: protection of a phage cocktail in a capsule including an anti-UV filter. A phage cocktail directed against Salmonella bacteria, Salmofresh™ marketed by Intralytix Inc., was encapsulated in capsules containing a UV filter. In parallel, two individual phages composing the said cocktail, respectively phage SBA 1781 and phage SPT, were encapsulated in the same type of capsules. All these phages were exposed to UV according to the protocol indicated above and counted, in comparison with the phage cocktail or the free phage and a negative control. This experiment was performed with both wet and dry capsules. The results are presented in Tables 11 and 12 below.

[0084] [Table 1 1] tps expo (min) control neg free phage CH El CH E2 0 LDD l,96E+05 2,80E+02 2,10E+02 10 LDD LDD 2.80E+02 l.70E+02 20 LDD LDD 7.00E+01 4.00E+01 30 LDD LDD 2.00E+01 l.00E+02 tps expo (min) control neg free phage CH El CH E2 0 LDD 2.10E+07 4.40E+05 3.60E+05 10 LDD LDD l,68E+05 l,78E+05 20 LDD LDD 2.41E+05 2.18E+05 30 LDD LDD l,27E+05 9,50E+04

[0085] [Table 12] 10 LDD l,40E+02 7,60E+04 2,30E+04 20 LDD LDD 3.60E+04 l.80E+04 30 LDD LDD 2.90E+04 3.90E+04

[0086] These results show a very significant decrease in the quantity of phages in the free phage control (i.e., non-encapsulated) after 10 minutes of UV exposure and viability preserved by the capsules comprising a UV filter. Said viability being demonstrated via the antibacterial activity of the phages against their target bacteria, these results demonstrate the stability of the antibacterial composition according to the invention.

Claims

Claims

1. [Antibacterial composition comprising: at least one bacteriophage selected from Podoviridae, Myoviridae and Siphoviridae, as active agent, in a liquid core, a gelled shell encapsulating the liquid core comprising said bacteriophage and comprising colloidal UV filter particles dispersed in which the UV filter is not a soluble antioxidant compound.

2. Antibacterial composition according to claim 1 in which the UV filter is chosen from titanium oxide, carbon black, biochar, charcoal, latex, silica, clays, and mixtures thereof.

3. Composition according to one of claims 1 or 2 in which the bacteriophage targets at least one plant pathogenic bacterium.

4. Composition according to claim 3 wherein said at least one plant pathogenic bacterium is selected from Erwinia spp., Xanthomonas spp., Xylella spp., Pseudomonas spp., Ralstonia spp.

5. A composition according to claim 4 wherein said plant pathogenic bacterium is of the genus Erwinia.

6. Composition according to one of claims 1 or 2 in which the bacteriophage targets at least one pathogenic bacterium capable of infecting humans, preferentially chosen from Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Staphylococcus aureus, Clostridium perfringens or a bacterium of the genus Salmonella.

7. Composition according to one of claims 1 or 2 in which the bacteriophage targets at least one animal pathogenic bacterium, preferentially chosen from Escherichia coli, Salmonella, Campylobacter, Staphylococcus, Flavobacterium, Yersinia, Clostridium.

8. Composition according to one of claims 1 or 2 in which the bacteriophage targets at least one pathogenic bacterium capable of contaminating products from the agri-food industry, said bacterium being preferentially chosen from Listeria, Salmonella, Shigella, Escherichia coli, Campylobacter jejuni.

9. Use of an antibacterial composition according to any one of claims 1 to 5 for the treatment of plant crops, their seeds or harvested products.

10. Use of an antibacterial composition according to any one of claims 1 to 5 for the treatment of soils. [Claim 1 1] A method for preventing and / or treating plant plants against at least one pathogenic bacterium comprising the steps of: providing an antibacterial composition comprising at least one bacteriophage as an active agent against said at least one pathogenic bacterium, said bacteriophage being protected against UV in the core of a microcapsule comprising colloidal UV filter particles dispersed in the shell encapsulating the core, in which the UV filter is not a soluble antioxidant compound, applying said composition to the aerial parts of the plant plant and / or by spreading on the soil, or around the seeds.

12. Use of an antibacterial composition according to one of claims 1, 2 or 6 for human nutrition and / or food.

13. Use of an antibacterial composition according to one of claims 1, 2 or 7 for animal nutrition and / or feed.

14. A pharmaceutical composition comprising an antibacterial composition according to one of claims 1 or 2.

15. Composition according to claim 14 for its use for the treatment of mammals and preferably humans.

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