FIELD

[0001] The present invention relates to the field of phage therapy for the treatment and control of bacterial infections, in particular infections caused by Salmonella.

BACKGROUND

[0002] The poultry industry has expanded around the world due to population growth, increased purchasing power and urbanization processes.

[0003] Breeding methods in the poultry industry have resulted in birds that respond to specialized purposes and are increasingly productive, causing the poultry industry to rapidly increase in size. However, this growth requires expert management in the development and transfer of feeding, slaughtering, processing, safety and efficiency technologies that will favor large-scale production chains.

[0004] One of the problems facing the poultry industry is poultry diseases caused by pathogenic organisms. Diseases affecting these birds can have repercussions that can be devastating for productivity, production and trade of live birds, meat and other poultry products. The pathogens that affect these birds are zoonotic and therefore can affect human health. One of these zoonotic pathogens is Salmonella.

[0005] The disease caused by Salmonella infection, salmonellosis, is the main cause of human infections through food. According to data provided by the European Union (EU), the origin of Salmonella is found mainly in animal species, particularly poultry. Infections in the poultry sector are related to Salmonella enterica serovars, the most prevalent being serovars Infantis, Agona, Senftenberg, Tennessee, among others. Due to the numerous Salmonella serovars, it is widely distributed in nature and there are differences in the dominant serotypes in each country and region. These particular characteristics lead to great difficulties for its prevention and control.

[0006] Conventional treatments to prevent Salmonella infection in poultry include vaccination, heat treatment of feed and/or incorporation of acids, frequent cleaning and disinfection, control and eradication of infestations such as mites and insects. In addition to not allowing the coexistence of other species or keeping them in separate places from the birds.

[0007] A treatment for Salmonella infection in birds is the use of specific bacteriophages against this bacterium. These biological agents aim to eliminate the pathogenic bacteria colonizing the intestine of these animals while remaining harmless to the microbiota of the birds and to eukaryotic cells.

[0008] The use of bacteriophages as a therapy attempts to decrease or eliminate the use and abuse of antibiotics and thereby resolve the infection without generating resistance in the bacteria. The distinctive features of phage therapy, related to their high specificity and self-replication capacity, make them especially attractive for preventing bacterial -related diseases, however, the narrow host range is a major constraint when finding phage candidates for therapies against Salmonella, because phages must be able to infect a wide range of different strains and serovars.

[0009] Phage cocktails may offer advantages over the use of individual phages, theoretically increasing the spectrum of bacterial lysis. The high variability in the natural epidemiology of Salmonella leads to phage cocktails that should comprise a large number of phages that allow a broad host range, where broad host range is understood as a formulation or cocktail of bacteriophages that possess lytic activity against 10 or more different serovars of the bacterium. However, the production of a high number of phages in a composition is laborious on an industrial scale. In addition high phage titers can cause adverse effects between the bacteriophages and even some of them have augmented instability in the formulation. [0010] The state of art fails to describe products that contain only few different bacteriophages but having a broad spectrum lytic activity that are capable of dealing with bacterial infections in uncontrolled environments, where the Salmonella population presents a large number of different serovars. It is urgent to develop new compositions of bacteriophages against Salmonella with a broad lysis spectrum that allow the control of diseases in the breeding, slaughter and processing stage in farm and/or hatchery animals, particularly in poultry.

SUMMARY

[0011] The present disclosure relates to the field of phage therapy for the treatment and control of bacterial infections, in particular infections caused by Salmonella.

[0012] In some aspects, the present disclosure provides a cocktail of bacteriophages exhibiting broad-spectrum lytic activity against pathogenic bacteria, particularly of the genus Salmonella, useful for the prevention, reduction and/or treatment of infections caused by these bacteria, an antibacterial formulation comprising said cocktail, the use of the antibacterial formulation in a method of prevention, reduction and/or treatment of diseases caused by bacteria such as Salmonella, a method of prevention, reduction and/or treatment of diseases caused by bacteria such as Salmonella and a method of disinfection of environments, surfaces and equipment for the decrease of the bacterial load of Salmonella.

[0013] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. INCORPORATION BY REFERENCE

[0014] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “figure” and “FIG.” herein), of which:

[0016] Figure 1 illustrates the effect of administration of an antibacterial formulation comprising bacteriophages M7, SAEN100P08, SAEN098P03 and SAENMB161P02 on the prevalence of Salmonella in naturally infected poultry. The presence of Salmonella was measured at day 28 of rearing in an untreated control group and in two treated groups.

[0017] Figure 2 illustrates the effect of administration of an antibacterial formulation comprising bacteriophages M7, SAEN100P08, SAEN098P03 and SAENMB161P02 on the average weight of naturally infected poultry. The average weight in grams of the birds (n=20) was measured at each week of life.

[0018] Figure 3 illustrates the effect of administration of an antibacterial formulation comprising bacteriophages L8, SAEN098P01 and SAEN098P03 on the load of Salmonella in naturally infected poultry. The load of Salmonella in poultry was analyzed through qPCR in the farms at days 28 and 42 of rearing in the control group and in the treatment group. DETAILED DESCRIPTION OF THE INVENTION

[0019] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0020] The present disclosure provides a cocktail containing only few different bacteriophages but having a broad- spectrum lytic activity against pathogenic bacteria, particularly of the genus Salmonella, useful for the prevention, reduction and/or treatment of infections caused by these bacteria. The present invention also relates to an antibacterial formulation comprising said cocktail for the prevention, reduction and/or treatment of infections caused by Salmonella', the use of said antibacterial formulation in a method of prevention, reduction and/or treatment of diseases caused by bacteria such as Salmonella-, the use of said antibacterial formulation for the manufacture of a medicament useful in the treatment of diseases caused by Salmonella-, a method of prevention, reduction and/or treatment of diseases caused by bacteria such as Salmonella; and, a method of disinfection of environments, surfaces and equipment for the decrease of the bacterial load of Salmonella.

[0021] The bacteriophages comprising this formulation were deposited with the International Depositary Authority of Canada (ID AC) in accordance with the Budapest Treaty for the deposit of biological samples.

[0022] The bacteriophages comprising the cocktail are identified as:

- Bacteriophage L8 (ID AC deposit N° 060820-01)

- Bacteriophage M7 (ID AC deposit N° 060820-06)

- Bacteriophage SAENMB161P02 (ID AC deposit N° 121022-01)

- Bacteriophage SAEN098P01 (IDAC deposit N° 220422-01)

- Bacteriophage SAEN098P03 (IDAC deposit N° 220422-02)

- Bacteriophage SAEN100P08 (IDAC deposit N° 220422-03). [0023] The bacteriophages disclosed in the present invention belong to the order of Caudovirales and all correspond to Salmonella-specific bacteriophages. The identification of the bacteriophages is set forth in their respective deposit certificate which was made under the Budapest Treaty. Said bacteriophages, when administered to animals, are useful for the control of different Salmonella serovars.

[0024] In a first embodiment, the present invention relates to a cocktail of bacteriophages with broad-spectrum lytic activity against pathogenic bacteria, particularly of the genus Salmonella, useful for the prevention, reduction and/or treatment of infections caused by these bacteria.

[0025] In one embodiment, the cocktail of the present invention comprises at least one of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, SAEN098P03 and SAEN100P08.

[0026] In another embodiment, the cocktail comprises at least two of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0027] In another embodiment, the cocktail comprises at least three of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0028] In another embodiment, the cocktail comprises at least four of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0029] In another embodiment, the cocktail comprises at least five of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0030] In another embodiment, the cocktail comprises the six bacteriophages with lytic activity against Salmonella L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08. [0031] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0032] The present disclosure provides a cocktail containing only few different bacteriophages but having a broad- spectrum lytic activity against pathogenic bacteria, particularly of the genus Salmonella, useful for the prevention, reduction and/or treatment of infections caused by these bacteria. The present invention also relates to an antibacterial formulation comprising said cocktail for the prevention, reduction and/or treatment of infections caused by Salmonella', the use of said antibacterial formulation in a method of prevention, reduction and/or treatment of diseases caused by bacteria such as Salmonella-, the use of said antibacterial formulation for the manufacture of a medicament useful in the treatment of diseases caused by Salmonella-, a method of prevention, reduction and/or treatment of diseases caused by bacteria such as Salmonella; and, a method of disinfection of environments, surfaces and equipment for the decrease of the bacterial load of Salmonella.

[0033] The bacteriophages comprising this formulation were deposited with the International Depositary Authority of Canada (ID AC) under the Budapest Treaty for the deposit of biological samples.

[0034] The bacteriophages comprising the cocktail are identified as:

Bacteriophage L8 (ID AC deposit N° 060820-01) Bacteriophage M7 (ID AC deposit N° 060820-06) Bacteriophage SAENMB161P02 (ID AC deposit N° 121022-01) Bacteriophage SAEN098P01 (IDAC deposit N° 220422-01) Bacteriophage SAEN098P03 (IDAC deposit N° 220422-02) Bacteriophage SAEN100P08 (IDAC deposit N° 220422-03).

[0035] The bacteriophages disclosed in the present invention belong to the order of Caudovirales and all correspond to Salmonella-specific bacteriophages. The identification of the bacteriophages is set forth in their respective deposit certificate. Said bacteriophages, when administered to animals, are useful for the control of different Salmonella serovars.

[0036] In a first embodiment, the present invention relates to a cocktail of bacteriophages with broad-spectrum lytic activity against pathogenic bacteria, particularly of the genus Salmonella, useful for the prevention, reduction and/or treatment of infections caused by these bacteria.

[0037] In one embodiment, the cocktail of the present invention comprises at least one of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, SAEN098P03 and SAEN100P08.

[0038] In another embodiment, the cocktail comprises at least two of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0039] In another embodiment, the cocktail comprises at least three of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0040] In another embodiment, the cocktail comprises at least four of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0041] In another embodiment, the cocktail comprises at least five of the bacteriophages with lytic activity against Salmonella wherein the bacteriophages are selected from L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08.

[0042] In another embodiment, the cocktail comprises the six bacteriophages with lytic activity against Salmonella L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08. [0043] In an optional embodiment, the cocktail further comprises selected bacteriophages of the order Caudovirales having specific lytic activity against bacteria of the Salmonella genus.

[0044] In a preferred embodiment, the bacteriophages comprising the cocktail are in the same proportion to each other. In another embodiment, the bacteriophages comprising the cocktail are in different proportions to each other. In a preferred embodiment, the bacteriophage cocktail comprises bacteriophages L8, M7, SAENMB161P02, SAEN098P01, and

SAEN098P03 and SAEN100P08 in the same proportions.

[0045] In one embodiment, the concentration of the bacteriophages in the cocktail is IxlO ⁴ to IxlO ¹² DNA copies/mL, preferably IxlO ⁶ to IxlO ⁹ DNA copies/mL.

[0046] The bacteriophages described in the present invention have not been previously described in the state of the art so that their use in a cocktail or in an antibacterial formulation comprising the cocktail, uses thereof and the method of application are presented as a new alternative that meets the objective of solving the technical problem described.

[0047] In a preferred embodiment, the bacteria of the genus Salmonella correspond to serovars Minnesota, Heidelberg, Mbandaka, Schwarzengrund, Senftenberg, Anatum, Newport, Agona, Tennessee, Thompson, Alachua, Braenderup, Infantis, Worthington, Havana, Cerro, Idikan, Livingstone, Ouakam, Rissen, Molade, Muenchen, Gallinarum, Give, Grumpensis, I l,4,[5],12:d:-, Saintpaul, Corvallis, I l,4,[5],12:i:-, Javiana, Montevideo, Muenster, Sandiego, Soerenga, Panama, Typhimurium, Gloucester, II 42:r:-, Albany, Brandenburg, Glostrup, Kentucky, Coeln, Meleagridis, Morehead, Saintpaul, Ohio, Oranienburg, Orion, Yoruba, Bredeney, Cubana, Gaminara, Hadar, Il ,4, [5], 12:r:-, I X:z4,z23:-, Isangi, IV T:z4,z23:-, Johannesburg, Kiambu, Lexington, Lille, London, Madelia, Oslo, Poona, Rubislaw, among others.

[0048] In another embodiment, the present invention relates to an antibacterial formulation comprising a cocktail of bacteriophages with broad-spectrum lytic activity against pathogenic bacteria, particularly of the genus Salmonella', a vehicle; a solvent, and/or veterinary and/or pharmaceutically acceptable excipients.

[0049] In the antibacterial formulation described as part of the scope of the invention, pH stabilizers, vehicles, solvents and veterinary and/or pharmaceutically acceptable excipients were selected.

[0050] The vehicle of the formulation described as part of the invention may comprise sterile liquids, selected from water and oils, including those of animal, vegetable or synthetic origin. In a preferred embodiment, aqueous solutions are employed as the vehicle, including sodium chloride.

[0051] The solvent of the formulation described as part of the invention may comprise sterile liquids. In a preferred embodiment, water or aqueous solutions are employed as solvent.

[0052] Included in the formulation are veterinary and/or pharmaceutically acceptable excipients corresponding to substances or compounds that give characteristics to the formulation that ensure the stability, bioavailability, acceptability and ease of administration of the active principle. Veterinary and pharmaceutically acceptable excipients of the formulation include preservatives selected from the ionic salts group, salts from the parabens group and salts from the chelators group.

[0053] Preservatives acceptable as excipients for the formulation include benzoic acid and benzoates, boric acid and borates, benzyl alcohol, cyclodextrins, benzalkonium chloride, fragrances, fructose, propylene glycol and propylene glycol esters, sodium, sodium salts (sorbates, organic salts such as sodium acetate, sodium butanoate, sodium benzoate, sodium hydroxybenzoate, parabens, sodium methyl-4-hydroxybenzoate, sodium stearate, sodium propionate, sodium propyl-4-hydroxybenzoate, sodium citrate, sodium lactate or sodium salts of organic acids. Also included are chelating preservatives such as trisodium ethylenediamine disuccinate acid, lactic acid, citric acid, ethylene diamine tetraacetic acid (EDTA), phytic acid, among other chelating agents acceptable for veterinary and pharmaceutical use. [0054] In one embodiment, in the antibacterial formulation of the present invention, the bacteriophages are in a proportion of 1:1000 to 1000:1 with the other components of the formulation (vehicle, buffer and/or veterinary and pharmaceutically acceptable excipients).

[0055] In another embodiment, the invention relates to an antibacterial formulation comprising a bacteriophage cocktail for veterinary use.

[0056] In a preferred embodiment, the antibacterial formulation is administered in solid, powder, liquid, aqueous suspension, aerosol, emulsifier or other form of administration.

[0057] In another preferred embodiment, the antibacterial formulation is administered as an additive in the feed or drinking water of the animals.

[0058] Preferably, the animals correspond to farm and/or hatchery animals in breeding, slaughter and processing stage, particularly poultry.

[0059] In another embodiment, the invention relates to an antibacterial formulation comprising a bacteriophage cocktail for use as a disinfectant for environments, surfaces and equipment.

[0060] In another embodiment, the present invention refers to the use of the antibacterial formulation in a method for the prevention, reduction, and/or treatment of infections caused by pathogenic bacteria, particularly of the genus Salmonella. Preferably, the animal in need of treatment corresponds to farm and/or hatchery animals in breeding, slaughter and processing stage, particularly poultry.

[0061] In another embodiment, the present invention refers to the use of the antibacterial formulation for the manufacture of a medicament useful in the treatment of infections caused by pathogenic bacteria, particularly of the genus Salmonella.

[0062] In another embodiment, the present invention refers to a method for the prevention, reduction, and/or treatment of infections caused by pathogenic bacteria, particularly of the genus Salmonella, wherein said method comprises administering a therapeutically effective amount of the antibacterial formulation comprising the cocktail to an animal in need of treatment. In particular, the administration of the antibacterial formulation is in the form of a suspension, injectable and/or orally administrable dosage, spray, aerosol, among others. Preferably, the animal in need of treatment corresponds to farm and/or hatchery animals in breeding, slaughter and processing stage, particularly poultry.

[0063] In another embodiment, the antibacterial formulation of the present invention is administered to the animal in need of treatment through any route of administration, such as oral, ocular, intramuscular, subcutaneous, parenteral, topical, intranasal or related routes.

[0064] In another embodiment, the present invention refers to a method of disinfecting environments, surfaces and equipment to decrease the bacterial load of bacteria of the genus Salmonella, wherein said method comprises: a) provide an effective amount of the antibacterial formulation according to the present invention; and, b) contact an environment, surface and/or equipment with said formulation.

[0065] As described, the cocktail of the present invention, as well as the formulation comprising it, is useful for preventing the growth of bacterial strains of the genus Salmonella in farm and/or farmed animals, particularly poultry, and in particular for preventing and decreasing the growth of different Salmonella serovars in animals. The formulation is useful to avoid the emergence of antibiotic resistant strains, avoid the use of chemical cleaning compounds and their residual accumulation.

[0066] The present invention refers to bacteriophages L8, M7, SAENMB161P02, SAEN098P01, and SAEN098P03 and SAEN100P08, which correspond to bacteriophages with a broad lytic spectrum against a wide variety of Salmonella isolates. The lytic activity of the bacteriophages L8, M7, SAENMB161P02, SAEN098P01, SAEN098P03 and SAEN100P08 comprising the cocktail was tested on 638 Salmonella isolates corresponding to 68 different serovars obtained from different industrial poultry was determined. Bacteriophages showed variable lytic activity on the 68 serovars tested.

[0067] The cocktail of the present invention possesses lytic activity against a wide variety of Salmonella serovars, including Minnesota, Heidelberg, Mbandaka, Schwarzengrund, Senftenberg, Anatum, Newport, Agona, Tennessee, Thompson, Alachua, Braenderup, Infantis, Worthington, Havana, Cerro, Idikan, Livingstone, Ouakam, Rissen, Molade, Muenchen, Gallinarum, Give, Grumpensis, I 1 ,4, [5], 12:d:-, Saintpaul, Corvallis, I l,4,[5],12:i:-, Javiana, Montevideo, Muenster, Sandiego, Soerenga, Panama, Typhimurium, Gloucester, II 42:r:-, Albany, Brandenburg, Glostrup, Kentucky, Coeln, Meleagridis, Morehead, Saintpaul, Ohio, Oranienburg, Orion, Yoruba, Bredeney, Cubana, Gaminara, Hadar, Il,4,[5],12:r:-, I X:z4,z23:-, Isangi, IV T:z4,z23:-, Johannesburg, Kiambu, Lexington, Lille, London, Madelia, Oslo, Poona, Rubislaw, among others.

[0068] The bacteriophages comprising the present invention are safe for their application and veterinary administration, since they correspond to bacteriophages with lytic activity, which do not present codifying sequences for virulence factors, integrases or antibiotic resistance. Additionally, the analyses allow establishing that there are no indications that support the probability of transduction of bacterial DNA by the bacteriophages. These characteristics demonstrate their safety for inclusion in a veterinary product. The bacteriophages that compose the formulation comply with what is requested in the general guidelines of genomic information described by the Food and Drug Administration of the United States for the approval of use of a bacteriophage mixture (Phillipson et al., 2018).

[0069] Moreover, all these characteristics make it possible to establish that the bacteriophages comprising the formulation proposed in the present invention are safe and suitable for use in phage therapy, and for environmental and surface application. [0070] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0071] EXAMPLES

[0072] Example 1. Obtaining Salmonella strains.

[0073] A total of 638 bacterial isolates were obtained from samples taken from different industrial poultry production farms, particularly broiler hatcheries.

[0074] The isolates were processed to obtain pure cultures. For this, a loop was introduced into the culture, inoculated with it in a tube with TSB and incubated for 3 hours. The result of the incubation was seeded on a plate with CHROMagar® Salmonella agar and incubated overnight. The next day, one CFU was recovered from the culture and inoculated into a tube with TSB and incubated for 18 hours. The resulting saturated culture was used to prepare a bacterial pellet by centrifugation for 10 minutes at 5,000 g.

[0075] Total bacterial DNA was purified from the bacterial pellet using the Purelink Genomic DNA kit (Thermo Fisher), following the manufacturer's recommendations. DNA quantification was performed by fluorimetry in the Qubit Fluorometer 3.0 kit (Thermo Fisher), with the reagents and parameters recommended by the manufacturer.

[0076] With the DNA obtained in the previous step, DNA libraries were prepared using the Nextera XT DNA library preparation and Nextera XT Index kits (Illumina), according to the manufacturer's recommendations. The size profile of the libraries was verified using the Fragment Analyzer kit (Agilent), according to the manufacturer's instructions.

[0077] Massively parallel sequencing of the libraries was performed using the NextSeq High Output Reagent Kit 300 Cycles (Illumina). Reads with quality value lower than 20 and those with similarity to sequencing adapters were discarded and a de novo assembly was obtained from each sequenced genome. Valid assemblies were analyzed by genoserotyping by traditional MLST, ribosomal MLST, and typing of coding sequences for genes that make up the core genome of Salmonella enterica. The serovar of each isolate and the allelic endowment matrix of the core genome were recorded.

[0078] The analyses identified the bacterial isolates as belonging to 68 different Salmonella serovars. The number of isolates per serovar and their prevalence are shown in Table 1.

[0079] Table 1. Identified Salmonella serovars from environmental samples.

[0080] Example 2. Characterization of viral genomes.

[0081] To characterize the viral genomes, different in silica analyses were performed to describe the type of viral DNA packaging, determine the viral DNA replication cycle, detect the presence of coding sequences for integrases, virulence factors and bacterial resistance genes, and establish the taxonomic identity of the bacteriophages.

[0082] The bacteriophages obtained correspond to the order Caudovirales, which are characterized by being tailed, double-stranded DNA bacteriophages. Bacteriophages L8, SAEN098P03, M7 and SAENMB161P02 belong to the Myoviridae family, while bacteriophage SAEN098P01 belongs to the Ackermannviridae family and bacteriophage SAEN100P08 belongs to the Terenviridae family.

[0083] The map of the bacteriophage genome shows that there are no integrases, virulence factors or resistance genes, which are necessary conditions for bacteriophages used in phage therapy.

[0084] Example 3. Determination of the in vitro lytic activity of individual bacteriophages on Salmonella strains.

[0085] Suspensions of bacteriophages L8, M7, SAEN098P01, SAEN098P03, SAENMB161P02 and SAEN100P08 were prepared in TSB medium. Each of the bacteriophage suspensions was inoculated separately with a culture or saturated with a reference strain of Salmonella. This inoculum was incubated for a period of 18 hours at 37°C with constant shaking at 200 rpm.

[0086] To purify the bacteriophages from the culture, it was centrifuged at 3,200 g for 5 min to separate bacteria and large particles. The supernatant was filtered using a 0.45 pm porosity polyethersulfone (PES) membrane.

[0087] To obtain the viral titer, 15 mL of TSB with agar was placed and incubated at room temperature for a period of 30 minutes. In parallel, 3 mL of TSB with agar was mixed with 1 mL of saturated culture of each of the 638 host bacterial isolates and 1 mL of TSB and poured onto the previously prepared TSB plate.

[0088] The TSB plates were inoculated with 5 pL of each bacteriophage and incubated at 37°C. After the incubation period, the lytic activity of bacteriophages was evaluated on a scale from 0 to 4. The results are illustrated in Table 2.

[0089] Table 2. Plate lysis assays of bacteriophage isolates on Salmonella serovars.

: Clear halo; 3: Non-insulated PFUs; 2: Insulated PFUs; 1: Opaque halo; 0: No activity; No measurement [0090] The results shown in Table 2 demonstrate that the bacteriophages of the invention are able to inhibit or decrease the growth of the 68 tested Salmonella serovars.

[0091] Example 4. Determination of the in vitro lytic activity of a bacteriophage cocktail on Salmonella serovars.

[0092] From the 638 Salmonella isolates, 87 representative isolates were chosen based on their abundance and based on previous reports in which some of them were difficult to eradicate using conventional solutions such as antibiotics (for example, serovars Heidelberg, Minnesota and Mbandaka). These isolates were challenged with a bacteriophage cocktail comprising L8, M7, SAENMB161P02, SAEN098P01, SAEN098P03 and SAEN100P08 bacteriophages. For this purpose, a culture of each bacterial isolate was mixed in TSB medium having an ODeoo=0.3, and diluted l:10 ⁵ with bacteriophage suspensions. Bacteriophages in the cocktail were mixed in equal amounts to a final concentration of IxlO ⁴ to IxlO ¹² DNA copies/mL per bacteriophage and a final bacteria concentration of IxlO ⁶ to IxlO ⁹ CFU/mL. The mixtures were added to a 96-well plate and incubated for 18 hours at 37°C using EPOCH spectrophotometer with continuous orbital shaking. ODeoo was recorded every 10 minutes. The antimicrobial effect of the mixture was determined by comparing the ratio of the area under the growth curve in the presence and absence of the bacteriophage. Null effect (N): inhibition up to 15% of growth. Partial inhibition (P): between 15% and 85%. Total inhibition (T): reduction over 85%.

[0093] The challenge obtained a variable efficacy among the 20 serovars identified, the results obtained are shown in Table 3.

[0094] Table 3. Efficacy of bacteriophage cocktail on Salmonella serovars.

[0095] Example 5. Evaluation of the efficacy of an antibacterial formulation comprising a cocktail of 4 bacteriophages in the reduction of Salmonella in naturally infected poultry.

[0096] The effect of oral administration of an antibacterial formulation comprising a bacteriophage cocktail comprising bacteriophages M7, SAEN100P08, SAEN098P03 and SAENMB161P02 on the prevalence and load of Salmonella in poultry and aviaries during the rearing process in poultry naturally infected with Salmonella was evaluated. The evaluation was carried out in industrial aviaries with a capacity of between 14,000 and 30,000 birds per rearing cycle.

[0097] All birds from 3 rearing cycles were selected. Individuals from the first rearing cycle were not administered with the formulation, while individuals from the remaining two rearing cycles were administered with 3 doses of a formulation comprising a bacteriophage cocktail with the bacteriophages M7, SAEN100P08, SAEN098P03 and SAENMB161P02 at a concentration of IxlO ⁴ to IxlO ¹² DNA copies/mL applied at different days of the rearing cycle in the drinker water supply at final dilution of 1%.

[0098] The prevalence and load of Salmonella in poultry was analyzed by taking footwear cover samples on day 28 of rearing. Feed conversion ratio, and mortality were analyzed by analyzing these parameters on day 28 of rearing and productive parameters was analyzed by analyzing average weight in grams on a weekly basis.

[0099] Bacteria were recovered from footwear covers by resuspension in TPA. The samples were pre-enriched in TPA to later use a 1 mL aliquot for molecular detection of Salmonella.

[00100] The results of prevalence of Salmonella (presence or absence), feed conversion ratio and mortality are given in Table 4.

[00101] Table 4. Prevalence of Salmonella, feed conversion ratio (FCR) and mortality in poultry pavilions (Pav).

P: Presence of Salmonella-, A: Absence of Salmonella-, No measurement

[00102] The results show that there are significant differences in the Salmonella prevalence between the treated conditions (7.14% of presence of Salmonella in cycle 2 and 0% of presence of Salmonella in cycle 3) and the control condition (21,43% of presence of Salmonella in cycle 1) (Figure 1). In addition, there is a 2% reduction in the average mortality rate in the pavilions.

[00103] The results of average weight are shown in Figure 2. At day 42 (week 6), the treated group was significantly heavier than the control group, by 230 grams and weight gain was significantly higher in the treated group in 7.5%.

[00104] Example 6. Evaluation of the efficacy of an antibacterial formulation comprising a cocktail of 3 bacteriophages in the reduction of Salmonella in naturally infected poultry.

[00105] The effect of oral administration of an antibacterial formulation comprising a bacteriophage cocktail comprising bacteriophages L8, SAEN098P01 and SAEN098P03 on the prevalence and load of Salmonella in poultry and aviaries during the rearing process in poultry naturally infected with Salmonella was evaluated. The evaluation was carried out in industrial aviaries with a capacity of between 14,000 and 30,000 birds per rearing cycle.

[00106] All birds from 4 rearing cycles were selected. Individuals from the first rearing cycle were not administered the formulation, while individuals from the remaining three rearing cycles were administered 3 doses of a formulation comprising a bacteriophage cocktail with the bacteriophages L8, SAEN098P01 and SAEN098P03 at a concentration of IxlO ⁴ to IxlO ¹² DNA copies/mL applied at different days of the rearing cycle in the drinker water supply at final dilution of 1%.

[00107] The load of Salmonella in poultry was analyzed through qPCR in the farms after receiving the whole treatment at days 28 and 42 of rearing.

[00108] The results of the load of Salmonella are given in Table 5.

[00109] Table 5. Load of Salmonella spp. showing the comparison at days 28 and 42 between the Control and the Treatment groups in terms of average load of bacteria in poultry farms. The numbers in each cell indicate the load of Salmonella in logs.

No measurement [00110] The results show that the treatment with the antibacterial formulation comprising 3 bacteriophages reduces between 1-5 logs the load of Salmonella in naturally infected (Figure 3). On day 28 the load of Salmonella spp decreased between 0.5 and 6 log of orders of magnitude in the 76% of the farms. On day 42, the results improved and showed a reduction of Salmonella between 1 and 5 logs in the 92% of the farms where the product was reduced on day 28.

[00111] Example 7. Evaluation of safety and efficacy of antimicrobial formulation comprising a bacteriophage cocktail in the reduction of Salmonella in poultry.

[00112] The effect of oral administration of an antibacterial formulation containing a bacteriophage cocktail comprising bacteriophages L8, M7, SAENMB161P02, SAEN098P01, SAEN098P03 and SAEN100P08 on the prevalence and load of Salmonella in poultry and aviaries, and on productive and health parameters during the rearing process in SaZmoneZZa-infected birds was evaluated.

[00113] The evaluation was carried out in 120 1-day-old male birds, distributed in experimental groups of 24 birds.

[00114] Experimental groups:

S/T Control: 24 birds not subjected to intervention.

Treated Control: 24 birds receiving 17 doses of the antibacterial formulation. S-TYP Control: 24 birds inoculated with Salmonella and not receiving antibacterial formulation.

S-TYP-PENTA: 24 birds inoculated with Salmonella receiving 5 doses of the antibacterial formulation. Moderate Regimen.

S-TYP-E2D: 24 birds inoculated with Salmonella receiving 17 doses of the antibacterial formulation. Intensive Regimen.

[00115] Groups inoculated with Salmonella were administered an aliquot of 0.05mL of a solution with a bacterial load of IxlO ⁸ CFU/mL by oral gavage on the third day of life. [00116] Salmonella spp. load was determined in stool, weekly, between day 7 and 35, and in cecum and crop contents at day 14 and 35. In addition, the tolerance of healthy non-infected birds to the regimen that delivers a higher number of doses (Treated Control) was evaluated by assessing the general health status of the animals, their mortality, daily weight gain and weekly feed consumption.

[00117] Evaluation of the safety of the antibacterial formulation:

[00118] In order to evaluate the safety of the formulation, clinical and zootechnical parameters were compared in the untreated control group (S/T Control) and the intensively treated group (Treated Control), which received a 17-dose administration regimen.

[00119] Animal health status:

[00120] The results of daily weight gain in the different groups analyzed are given in Table 6.

[00121] Table 6. Calculated daily weight gain for study days 1-14, 14-35 and 1-35.

[00122] Daily weight gain was homogeneous among the different groups, with higher gains in the Treated Control and S-TYP-PENTA groups.

[00123] The results of total feed consumption per bird in the different groups analyzed are shown in Table 7.

[00124] Table 7. Feed consumption

[00125] No differences were observed between feed consumption in the different groups.

[00126] In addition, the necropsy of the birds did not reveal the presence of lesions or pathological changes in any of the organs evaluated. Regarding the productive performance, an increase of 209.5 g in body weight was observed in the birds belonging to the Treated group compared to the S/T Control.

[00127] No alterations in health status of animals were observed during the study and mortality remained at 0%.

[00128] Overall, the data indicate that the antibacterial formulation is safe and does not suggest health risks to the birds.

[00129] Evaluation of the efficacy of the antibacterial formulation:

[00130] In order to evaluate the efficacy of the formulation, the concentration of Salmonella in crop, feces and cecum was compared in the different study groups.

[00131] Salmonella spp. concentration:

[00132] Salmonella spp. were not detected in crop, cecum or feces samples from the S/T Control and Treated Control groups on any of the days evaluated during the study, indicating that the birds did not possess an endogenous population of Salmonella spp.

[00133] Salmonella concentration in crop:

[00134] The results of Salmonella concentration in crop in the different groups challenged with the bacteria are given in Table 8.

[00135] Table 8. Summary of the results obtained in the quantification of Salmonella in crop of each group at day 14 and 35. The results are shown as the median of the logarithm in base 10 of the concentration in MPN/g units.

[00136] A statistically significant decrease in the concentration of Salmonella in the crop at day 14 was observed in the moderate and intensive regimen with respect to the results of the control group (2.46 logio and 4.33 logio respectively). At day 35, a statistically significant decrease of 1.73 logio was observed in the intensive regime compared to the control group.

[00137] Salmonella concentration in the cecum:

[00138] The results of Salmonella concentration in cecum in the different groups challenged with the bacteria are given in Table 9.

[00139] Table 9. Summary of the results obtained in the quantification of Salmonella in the cecum of each group at day 14 and 35. The results are shown as the median of the logarithm in base 10 of the concentration in MPN/g units.

[00140] A statistically significant decrease in the concentration of Salmonella in the cecum was observed at day 14 in the moderate and intensive regimen with respect to the results of the control group (1.48 logio and 2.92 logio respectively). At day 35, a significant decrease was also observed in the treated groups (5.34 logio and 6.20 logio respectively).

[00141] Salmonella concentration in feces:

[00142] The results of Salmonella concentration in feces in the different groups challenged with the bacteria are given in Table 10.

[00143] Table 10. Summary of the results obtained in the quantification of Salmonella in feces of each group at day 7, 14, 21 and 35. The results are shown as the median of the logarithm in base 10 of the concentration in MPN/g units.

[00144] A statistically significant decrease of 2.81 logio at day 21 and 6.01 logio at day 35 in the mean bacterial load was observed with respect to the control group.

[00145] Overall, the data indicate that the antibacterial formulation is effective in reducing the Salmonella load in the crop, cecum and gullet, being effective for use in poultry flocks.