WO2023152549A1

WO2023152549A1 - Compositions for the treatment of e. coli and salmonella

Info

Publication number: WO2023152549A1
Application number: PCT/IB2022/051257
Authority: WO
Inventors: Nicolás FERREIRA SOTO; Hans PIERINGER CASTRO; Pablo CIFUENTES PALMA; Diego BELMAR ZAMBRANO
Original assignee: Phagelab Chile Spa
Priority date: 2022-02-11
Filing date: 2022-02-11
Publication date: 2023-08-17

Abstract

The present disclosure relates to a composition comprising a mixture of at least two bacteriophages, which are useful for combating the growth of Escherichia coli STEC and ETEC, and of different serovars of Salmonella, in farm animals suffering infections by these bacteria; processes for producing said compositions; stabilized formulations comprising said bacteriophages; and uses in the control of enteropathogenic bacteria in animals.

Description

COMPOSITIONS FOR THE TREATMENT OF E. COLI AND SALMONELLA

TECHNICAL FIELD

[1] The present disclosure may be applied in the field of veterinary compositions for combating the growth of pathogens in the intensive breeding of animals. Examples of animals include, but are not limited to, cattle, goats or sheep. The disclosure also presents, in some embodiments, a composition comprising a mixture of bacteriophages which may be useful for combating the growth of Shiga toxin-producing (STEC) and enterotoxigenic (ETEC) Escherichia coli bacteria, and different serovars of Salmonella enterica. In one aspect, the compositions of the present disclosure can be used in breeding animals. The disclosure also presents, in some embodiments, a production process, formulations comprising said bacteriophages, and uses for the control of enteropathogenic bacteria in animals.

BACKGROUND

[2] A significant number of livestock animals suffer from diarrhea during their first month of life, especially during the first week of life, in a condition referred to as neonatal diarrhea. Diarrheal episodes occur because not all animals consume colostrum at birth in adequate quantities, and they therefore do not receive important antibodies for defense against various pathogens. In calves, the clinical manifestation of diarrhea is varied and can range from mild diarrhea without systemic manifestations to extremely aggressive diarrhea associated with rapid dehydration. Such aggressive diarrhea may cause a variation in the acidbase relationship and electrolyte balance, ultimately leading to death of the animal within 12 hours. Even animals who survive these diarrheal episodes during their first days of life have an impact on the industry: the animals’ decreased fattening rate causes them to produce less meat or milk, thereby affecting production yields.

[3] The main pathogens causing such diarrhea may include Escherichia coli and Salmonella enterica. Diarrhea in animals and humans may be characterized by the presence of one or different pathotypes of E. coli, including: (1 ) enterotoxigenic E. coli (ETEC); (2) enteropathogenic E. coli (EPEC); (3) enteroinvasive E. coli (EIEC); (4) enterohemorrhagic E. coli (EHEC); (5) enteroaggregative E. coli (EAEC), (6) enteroadherent E. coli (EAdEC) and (7) vero toxin-producing or Shiga-\\ke toxin-producing E. coli (VTEC or STEC).

SUMMARY

[4] Recognized herein is a need for developing products that control the growth of relevant pathogens in livestock animals, specifically: (1 ) E. coli ETEC, (2) E. coli STEC, (3) Salmonella Infantis, (4) Salmonella Dublin, (5) Salmonella Typhimurium, (6) Salmonella Mbandaka, (7) Salmonella Anatum and (8) Salmonella Panama.

[5] Bacteriophages or phages are viruses that specifically infect and lyse bacteria and are composed mainly of genetic material and proteins. Phages have the ability to recognize the surface of bacterial cells with high specificity, inject their DNA or RNA, multiply inside the bacterium, where they then lyse it and release their progeny.

[6] Regarding phage replication, these can be classified in two ways: the lysogenic phase and the lytic phase. In the lysogenic or temperate cycle, phage DNA is integrated into the bacterial chromosome. This DNA replicates as part of the bacterial or host genome and can remain in a state of latency or prophage for long periods. On the other hand, in the lytic or virulent cycle, the phage binds specifically to its receptor on the surface of the bacterium, and then injects its genome into the bacterium. The genome inside the bacterium replicates using the enzymatic machinery of the bacterium, resulting in the assembly of new viral particles, which are finally released by lysis of the bacterium.

[7] For the use of phages in therapeutic or biocontrol applications, bacteriophages may correspond to those with lytic replication characteristics and adequate specificity for their application in different breeding animals, due to their rapid elimination of the host.

[8] In addition, it is important to highlight that, unlike current treatments to control pathogens in intensive animal breeding industries, bacteriophages do not generate antibiotic resistance and can eliminate bacteria that are multi-antibiotic resistant, thus contributing to generate a more environmentally friendly pathogen control strategy.

[9] With regards to E. coli, the main agent of calf diarrhea is the ETEC pathotype. Additionally, the STEC pathotype may cause diarrheal episodes in humans (Kolenda, Burdukiewicz and Schierack. 2015. A systematic review and meta-analysis of the epidemiology of pathogenic Escherichia coli of calves and the role of calves as reservoirs for human pathogenic E. coli. Front Cell Infect Microbiol. 5:23). ETEC pathotypes are characterized by their expression of thermostable toxins STa and STb and thermo labile toxins LT-I and LT-IL One of the ways to determine whether a biological sample has E. coli ETEC is to determine the presence of the coding gene for these toxins. On the other hand, STEC pathotypes are characterized by the presence of the toxins Stx1 and/or Stx2. Determination of the presence of genes coding for at least one of the two toxins is useful for determining whether a biological sample is positive for this pathotype. Due to the possible heterogeneity of pathotypes that can be found in different calf herds with scours, another type of classification is often used which is based on serotyping of O (lipopolysaccharide) and H (flagellum) antigens.

[10] The E. coli serotypes involved in episodes of neonatal diarrhea may include: (1 ) 044, 055, 0146, 01 13, 0121 , 026, 091 , 011 1 , 08, 0127, 086 and 0128; (2) 026, 0103 and 0146; (3) 0157, 026, 045, 0103, 01 1 1 , 0121 , and 0145; and (4) 025, 078, 086, 01 19, 0158, 0164, and 0157 (Aref, et al. 2018. Clinical and sero-molecular characterization of Escherichia coli with emphasis on hybrid strain in healthy and diarrheic neonatal calves in Egypt. Open Veterinary Journal, (2018), Vol. 8(4): 351 -359); Ndegwa., etal. 2020. Age-related differences in phylogenetic diversity, prevalence of Shiga toxins, Intimin, hemolysin genes and selected serogroups of Escherichia, coli from grazing meat goats detected in a longitudinal cohort study. BMC Veterinary Research volume 16, article number: 266); Browne., etal. 2018. Molecular epidemiology of Shiga toxin-producing Escherichia coli (STEC) on New Zealand dairy farms: application of a culture-independent assay and whole genome sequencing. Applied and Environmental Microb 84(14): e00481-18; Osman., et al. 2013. The Distribution of Escherichia coli Serovars, Virulence Genes, Gene Association and Combinations and Virulence Genes Encoding Serotypes in Pathogenic E. coli Recovered from Diarrhoeic Calves, Sheep and Goat. Transboundary and Emerging Diseases. 60: 69-78).

[11] Among these E. coli serotypes, one of the most relevant in calves with diarrhea is O157:H7 (Kang S., et al. 2004. Occurrence and characteristics of enterohemorrhagic Escherichia coli 0157 in calves associated with diarrhoea. Veterinary Microbiology 98(3-4): 323-328). This STEC strain is of great importance worldwide since it is also the main cause of bloody diarrhea in humans resulting from the consumption of contaminated food (Laegreid W. W., Elder R.O and Keen J.E. 1999. Prevalence of Escherichia coll 0157:1-17 in range beef calves at weaning. Epidemiol. Infect., 123 (1999), pp. 291 -298).

[12] As aforementioned, another pathogen that may be involved in diarrhea in livestock animals is Salmonella enterica. This bacterium generates significant economic losses in calves due to high mortality in young animals and decrease in weight (Rushton. 2009. The Economics of Animal Health and Production. CABI International, Cambridge, MA).

[13] Within the S. enterica species, about 2,500 serovars have been identified, but studies analyzing the prevalence of this bacterium in calves with neonatal diarrhea have determined that the most important serovars in decreasing order of importance include: (1 ) Dublin, (2) Cerro, (3) Newport, (4) Montevideo, (5) Kentucky, and (6) Typhimurium (Holschbach and Peek. 2018. Salmonella in Dairy Cattle. Veterinary Clinics: Food Animal Practice. 34(1): 133-154).

[14] There are other relevant Salmonella serovars that may cause neonatal diarrhea in calves, including: Mbandaka (McConnel., et al. 2019. Elucidating dairy calf mortality phenotypes through postmortem analysis. Journal of Dairy Science, 102 (5): 4415-4426), Panama (Molossi., et al. 2021. Epidemiological and pathological aspects of salmonellosis in cattle in southern Brazil. Pathology- Cienc. Rural 51 (3). https://doi.org/10.1590/0103- 8478cr20200459) and Anatum (Bilbao, etal. 2019. Detecton de serovares de Salmonella en terneros de crianza artificial de la region lechera Mar y Sierras, Argentina. Revista Argentina de Microbiologfa, 51(3), 241-246).

[15] Additionally, it has been determined that one of the main causative agents of gastroenteritis in children is Salmonella Infantis. Although its prevalence is not high in cattle herds, it is necessary to develop biological agents to control the growth of this serovar.

[16] Applicant has identified that there exist no stable solid formulations comprising a cocktail of bacteriophages against STEC and ETEC strains of E. coli and against different serovars of S. enterica, which are identical to the formulations of the present disclosure for use to prevent and/or treat colibacillosis or salmonellosis, in cattle, preferably in calves.

[17] The present disclosure specifically addresses this problem since the developed bacteriophage formulation is specific for different relevant serogroups of STEC and ETEC bacteria, as well as the most relevant worldwide Salmonella serovars.

[18] The present disclosure provides a composition comprising a mixture of at least two bacteriophages, which are useful for combating the growth of Escherichia coli STEC and ETEC, and of different Salmonella serovars, in breeding animals suffering infections by these bacteria. In particular, the present disclosure provides a composition comprising at least two bacteriophages having activity against strains of E. coli STEC and ETEC and against at least five different serovars of Salmonella.

[19] In addition, the present disclosure provides processes for producing the compositions, stabilized formulations comprising said bacteriophages, and to uses in the control of enteropathogenic bacteria in animals.

[20] 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

[21] 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.

DESCRIPTION OF THE FIGURES

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

[23] Figure 1 . Antibiotic resistance profiles of E. coli isolates obtained from calves.

[24] Figure 2. Antibiotic resistance profiles of Salmonella spp. isolates obtained from calves.

[25] Figure 3. Duration time of the first episode of diarrhea in calves with or without continuous addition of bacteriophages in the liquid diet (n=96).

[26] Figure 4. Duration of diarrhea episodes during the first 30 days of life of calves with or without continuous addition of bacteriophages in liquid diet (n=96).

[27] Figure 5. Average daily weight gain (DWG) in calves in the neonatal period (30 days of life) with or without continuous addition of bacteriophages in liquid diet (n=175).

[28] Figure 6. Average daily weight gain (DWG) in calves with partial weaning (42 days) and total weaning (80 days), with or without the addition of bacteriophages continuously in milk (n=175).

[29] Figure 7. Effect of the formulation of the disclosure on the severity of diarrhea in calves at days 0, 7, 24 and 70.

[30] Figure 8. Average weight gain (kg) in calves with or without the addition of bacteriophages at 0, 7, 24 and 70 days.

[31] Figure 9. Average daily weight gain (g/day) in calves with or without addition of bacteriophages at 0-7 days, 0-24 days and 0-70 days.

DETAILED DESCRIPTION

[32] While various embodiments of the disclosure 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 disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed.

[33] The present disclosure provides an antibacterial formulation comprising an effective amount of at least two bacteriophages exhibiting specific lytic activity against strains of enteropathogens such as E. coli STEC and ETEC and comprising an effective amount of one or more bacteriophages exhibiting lytic activity against different Salmonella serovars. Antibacterial formulations of the present disclosure may be used with specificity against various pathotypes of E. coli and Salmonella. Such formulations may be used for various applications, such as veterinary use. In some instances, veterinary use may be the treatment or prevention of neonatal diarrhea in cattle.

[34] More specifically, the present disclosure relates to a solid antibacterial formulation for veterinary use comprising a mixture of one or more bacteriophages specific against E. coli STEC and ETEC and comprising an effective amount of at least two bacteriophages exhibiting lytic activity against different Salmonella serovars, together with a pharmaceutically and veterinary acceptable excipients.

[35] Bacteriophages comprising the formulation were identified and deposited with the International Depositary Authority of Canada (IDAC) and the American Type Culture Collection (ATCC, USA) in accordance with the provisions for a deposit under the Budapest Treaty. As set out in the original deposit certificates, the bacteriophages comprising the composition of the present disclosure are identified as:

PHT-EC-B5 Bacteriophage* (ATCC deposit PTA-122323)

EcoM-FR3 Bacteriophage (IDAC deposit 121121 -08)

EcoM-FR5 Bacteriophage (IDAC deposit 200520-01)

SenM-L8 Bacteriophage (IDAC deposit 060820-01)

SenM-STM1 Bacteriophage (IDAC deposit 060820-03)

SenS-STM47B Bacteriophage (IDAC deposit 060820-05)

[36] *For purposes of the present disclosure, the PHT-EC-B5 bacteriophage may also be referred to as EcoM-D bacteriophage.

[37] It is an object of the present disclosure to provide an effective and safe antibacterial formulation for treating infections caused by Escherichia coli STEC and ETEC, such as colibacillosis, and at the same time infections caused by Salmonella, in non-human animals.

[38] Bacteriophages that compose this formulation have not been previously disclosed in the state of the art. Therefore, the formulation and the method to administer it constitute new and effective alternatives to meet the proposed objectives.

[39] The disclosure also presents the use of the described antibacterial formulation as a medicament for the prevention and treatment of infectious diseases caused by Escherichia coli STEC and ETEC as well as various Salmonella serovars. Examples of such infectious diseases include coli baci llosis and salmonellosis.

[40] The disclosure also presents an antibacterial formulation capable of preventing the emergence of antibiotic resistant bacteria and the accumulation of residual antibiotic in animals when treating infections caused by Escherichia coli STEC and ETEC, such as colibaci llosis, with conventional methods.

[41] The disclosure also presents an antibacterial formulation capable of preventing the emergence of antibiotic resistant bacteria and the accumulation of residual antibiotic in animals when treating infections caused by different Salmonella serovars, such as salmonellosis, with conventional methods. In some instances, the Salmonella serovars targeted by the bacteriophages of the present disclosure are selected from Salmonella Infantis, Salmonella Typhimurium, Salmonella Mbandaka, Salmonella Anatum, Salmonella Dublin, and Salmonella Panama.

[42] The antibacterial formulation may be administered to the animal orally, in format or presentation as a liquid or powder.

[43] The antibacterial formulation may be provided in a powder formulation for quickly and easily applying the bacteriophage composition to a feed for animal consumption. The animal may be a breeding animal or livestock animal. Examples of such animals may include cattle, sheep and goat.

[44] The antibacterial formulation may be created from the microencapsulation of the mixture or "cocktail" of bacteriophages in a matrix that may consist of an ingredient selected from Molasses, Corn, Soy, Wheat, Rice, Barley or Rye, among others; an additive selected from Flavorings, Colorings, Preservatives, Antioxidants, Acidulants, Sweeteners, Thickeners, Starch Derivatives, Flavorings or Emulsifiers, among others; or a selected complement of vitamins, minerals, amino acids, essential fatty acids, fiber or herbal extracts, among others to a combination thereof, by means of a conventional drying method which may be selected from the group consisting of dehydration, freeze drying, atomization or a combination thereof, thus allowing to obtain a powder formulation and that the matrix comprises bacteriophages, thus being able to be added to other preparations or complex foods fed to non-human animals.

[45] Antibacterial formulations as described herein may be manufactured by the following process:

A. Each bacteriophage is propagated separately with its respective host bacterium using standard culture conditions;

B. After the end of the incubation period the bacteriophages are purified from the culture medium by centrifugation and use of 0.22 pm filters;

C. Each bacteriophage formulation is quantified in terms of the number of PFU/mL obtained by standard methodologies;

D. At least two of the six individual formulations obtained in the previous steps are mixed: SenM-L8 bacteriophage (IDAC deposit 060820-01), SenM-M7 bacteriophage (IDAC deposit 060820-06), SenS-STM47B bacteriophage (IDAC deposit 060820-05), PHT-EC-B5 bacteriophage (ATCC deposit PTA-122323), EcoM-FR3 bacteriophage (IDAC deposit 121 121 -08) and EcoM-FR5 bacteriophage (IDAC deposit 200520-01 ). In this step, appropriate amounts of each of the individual formulations are added so that each of the bacteriophages is left at a final concentration of between 1 x104 to 1 x1010 PFU/mL;

E. The formulation obtained in step D. is combined with a food matrix in a ratio between 2-10 of bacteriophages mixtures and between 20 and 70% w/v food matrix;

F. Add a chelating agent, or one of its salts, preferably EDTA, in a proportion between 0.1 % and 0.3% w/v in relation to the amount of the mixture obtained in step E.;

G. Add water in a proportion of at least between 40 and 80 %v/v in relation to the composition obtained in step F.;

H. To mix the mixture obtained in G. for at least 24 hours, until obtaining homogeneity; and

I. Dry the sample in a suitable industrial equipment until obtaining a solid product with a humidity between 0,001 % and 10 %.

[46] As a skilled person will note, the water used in the production process could be more or less than 65 %v/v, since water serves as a diluent to ensure homogenization of the bacteriophages in the food matrix. Therefore, this ratio should not be considered as limiting, but a person skilled in the art could reproduce the process by adding more or less water, but adding more water would only lengthen the drying process.

[47] Similarly, the food matrix could be added in other proportions such as in the range of 20 %w/v to 50 %w/v.

[48] In the antibacterial formulation described as part of the scope of the disclosure, the bacteriophages are in the formulation in amounts or concentrations of 1 x106-1 x109 PFU/mL, preferably 1 x10⁷-1 x10⁸ PFU/mL. In a preferred embodiment of the disclosure the bacteriophages are in amounts or concentrations of 5x10⁷-5x10⁸ PFU/mL.

[49] In the antibacterial formulation described as part of the scope of the disclosure, pharmaceutically and veterinary acceptable matrixes, chelators and excipients were selected.

[50] In the antibacterial formulation described as part of the scope of the disclosure, the bacteriophages and the matrix used, are in a ratio between 2-10 of bacteriophages mixtures and between 20 and 70% w/v, respectively.

[51] In a further preferred embodiment, the formulation comprises EDTA or its disodium salt in a ratio between 0.1 % and 0.3% w/v.

[52] In the antibacterial formulation described as part of the scope of the disclosure, the bacteriophages, food matrix and chelators are preferably in a ratio of 6:64:0.17, respectively, and water to make up 100%.

[53] The dry formulation obtained by the drying process described above is then ready to be used by mixing it with a food or beverage that can be administered orally to the non-human animal, thus reducing the complexity, variability and risk of adverse effects of techniques such as intravenous, subdermal, gastric and/or nasogastric tube administration, among others.

[54] When the dried formulation is added to another food or beverage, for example milk replacer or water that is directly delivered orally to non-human animals, the mixture of bacteriophages is incorporated into the entire digestive tract of the animal, acting if and only if they encounter any of the target bacteria, initiating only at that moment their lytic cycle.

[55] Bacteriophages composing the antibacterial formulation may be safe for veterinary application and administration. This is because they correspond with lytic activity, which does not present coding sequences for virulence factors, integrases or antibiotic resistance. Additionally, there are no indications supporting the probability of transduction of bacterial DNA by the phages. These characteristics demonstrate their safety for inclusion in a veterinary product, as demonstrated throughout this application. Bacteriophages that compose formulations as described herein comply with the general guidelines of genomic information described by the Food and Drug Administration of the United States for the approval of the use of a bacteriophage mixture.

[56] The disclosure also provides a method for preventing or treating infectious diseases caused by Escherichia co// STEC and ETEC, such as colibacillosis, and at the same time treating infectious diseases caused by Salmonella serovars Infantis, Typhimurium, Mbandaka, Anatum, Dublin and Panama, wherein said method comprises administering the described antibacterial formulation in a non-human animal. The non-human animal may be a breeding animal or cattle, a sheep, or agoat. The disclosure also provides a method comprising administering the antibacterial formulation comprising the bacteriophages to a non-human animal orally, wherein the non-human animal is preferably a calf.

[57] The disclosure further provides a method that may comprise oral administration of any antibacterial formulation of this disclosure to neonatal calves aged 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days. The method may also comprise the oral administration of any antibacterial formulation of this disclosure to neonatal calves on days 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60 or more days after birth. The method may comprise oral administration of any antibacterial formulation of this disclosure to neonatal calves on day 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the neonatal calf’s life.

[58] Days 1 through 7 of a calf’s life are associated with the highest rate of neonatal diarrhea caused by E. coli. The disclosure further provides a method that may comprise oral administration of any antibacterial formulation of this disclosure to neonatal calves aged 0, 1 , 2, 3, 4, 5, 6, 7, or more days. The method may also comprise the oral administration of any antibacterial formulation of this disclosure to neonatal calves on days 0, 1 , 2, 3, 4, 5, 6, 7, or more days after birth. The method may comprise oral administration of any antibacterial formulation of this disclosure to neonatal calves on day 0, 1 , 2, 3, 4, 5, 6, or 7 of the neonatal calf’s life.

[59] Days 5 through 30 of a calf’s life are associated with the highest rate of neonatal diarrhea caused by Salmonella. The disclosure further provides a method that may comprise oral administration of any antibacterial formulation of this disclosure to neonatal calves aged 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days. The method may also comprise the oral administration of any antibacterial formulation of this disclosure to neonatal calves on days 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more days after birth. The method may comprise oral administration of any antibacterial formulation of this disclosure to neonatal calves on day 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 of the neonatal calf’s life.

[60] 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 i nvention 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.

DEFINITIONS

[61] The following are definitions of terms that enable a complete understanding of this disclosure. The definitions comprise clear scientific and technical terms. Any changes in these definitions will be indicated in the text.

[62] The term "bacteriophage" or "phage" refers to a type of virus whose host cells are bacteria. For the purposes of the disclosure, the host bacteria correspond to one or more strains of Salmonella spp. or to one or more strains of E. coli ETEC and STEC. For the purposes of the disclosure, the term may also be used to refer to fragments of such viruses or assemblies including these parts, whose functional activity is similar to that of using them in their entirety.

[63] The term "lytic activity" refers to the property of a virus to cause lysis in its host cell.

[64] The term "phage therapy" refers to the use of bacteriophages to treat a bacterial infection, using those that are specific for that infection.

[65] The term "mix" or "mixture" or "combination" of phages refers to a mixture containing at least two different bacteriophages, which constitutes the active ingredient of the antibacterial formulation.

[66] The term "antibacterial formulation" refers to a composition that is directed to the prevention or treatment of infections caused by bacteria. For the purposes of the disclosure, "antibacterial" is understood as the total elimination or decrease or reduction of the bacterial population or bacterial load.

[67] The unit "PFU/mL (Plaque Forming Unit in English) or UFP/mL (Lysis Plate Forming Units)" is a measure of the number of lysis halos present on a bacterial culture plate per unit of virus volume, where theoretically each halo is formed by the presence of a single virus. In this case, it is a unit for quantifying the number of phage viral particles capable of lysing host cells.

[68] The term "bacterial infection" refers to the invasion of these pathogenic microorganisms into a host, resulting in disease.

[69] The term "serovar" or "serotype" refers to a group of bacterial species that share functional structures on their surface (antigens), which allow them to infect their host cells and trigger pathogenesis.

[70] The term "effective amount", for the purposes of the disclosure, refers to an adequate concentration of the bacteriophage or bacteriophages comprising the antibacterial formulation to make the treatment of the disease. This "effective amount" may vary according to the bacterial strain to be targeted, the subject to be administered, or the type of formulation to be prepared.

[71] The term "veterinary acceptable vehicle or excipient" refers to any component, regardless of its nature, that allows the correct administration of the bacteriophages in the species to be treated. Examples of veterinary acceptable excipients are chelating salts, matrices such as maltodextrin, pH stabilizers, among others.

[72] In the case of a pH stabilizer, this corresponds to a substance, compound or mixture of compounds that have the ability to maintain a constant pH when small amounts of acids or bases are added.

[73] The term "vehicle" refers to a diluent, adjuvant or excipient with which the active ingredient is administered. Such pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of animal, vegetable or synthetic origin. Water or saline aqueous solutions are preferably used as a vehicle.

[74] The term "veterinary use" refers to its application only in non-human animals.

[75] The term "treatment" or "treat" and its derivatives refers to the care and combating of a disease or the symptoms caused by it. For the purposes of the disclosure, "treatment" is understood as the administration of the formulation in order to eliminate, stabilize or ameliorate the symptoms of the illness, or to kill or reduce the bacterial population causing the disease. [76] For the purposes of the disclosure, when referring to "non-human animal" these correspond to farm animals or livestock, including but not limited to cattle, sheep and goats.

[77] For their part, the terms "prevention" or "prevent" and its derivatives refer to reducing the likelihood of contracting a disease. In this case, it refers to decreasing or avoiding the spread of a bacterial infection by administering the formulation.

EXAMPLES

[78] Example 1 : Isolation and Characterization of Bacteriophage and Bacterial strains.

[79] 154 feces samples were taken from milk producing calves from 47 different production farms located in Chile, Argentina and Brazil. Sterile swabs were used for sample collection, the transport medium was Stuart medium and the samples were stored at 4°C unti I processing.

[80] Bacteria and phages present in the samples were isolated by methods well known in the art (Sambrook, Joseph. 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press), which is entirely incorporated herein by reference.

[81] Identification of E. co// strains

[82] For the isolation of pathogenic E. coli, selective media such as CHROMAGAR® were used and the confirmation of these isolates was performed by qPCR for the UidA 405, STa, STb and stx1 genes.

[83] In addition, Escherichia coli strains were acquired from the ISP repository (Institute of Public Health, Chile) and the American Type Culture Collection (ATCC, USA) (Table 1).

Table 1 : Escherichia co// strains acquired from ISP (Chile) and ATCC (USA).

[84] Identification of Salmonella enterica strains

[85] The detection of Salmonella enterica strains was performed according to the VIDAS® EASY SLM AFNOR BIO 12/16-09/05 Screening Method or Method for detection of motile Salmonella spp. in feces and crawling slurry according to ISO 6579:2002/Amd. Confirmation of Salmonella isolates was performed by qPCR.

[86] In addition, Salmonella enterica strains were acquired from the ISP repository (Institute de Salud Publica, Chile) and the American Type Culture Collection (ATCC, USA) (Table 2).

Table 2. Salmonella enterica strains acquired from ISP (Chile) and ATCC (USA).

[87] Of those samples that were positive for the presence of Salmonella, a saturated culture was prepared from these and inoculated into a tube containing 5 mL of TSB which was incubated overnight at 37°C. After the incubation period, these saturated cultures were seeded on ChromID Salmonella agar (Biomerioux), XLD agar, or DMLIA agar, incubated, and then the colonies were isolated and stored according to the manufacturer's instructions.

[88] The results obtained are summarized in Table 3.

Table 3. Summary of strains identified as Salmonella and as pathotypes of E. coll in the analyzed samples .

[89] Antibiotic resistance analysis

[90] Then, it was analyzed whether the isolated bacteria were resistant to antibiotics, and as shown in Figure 1 (for E. coli) and Figure 2 (for Salmonella), a large percentage of the bacterial isolates obtained showed resistance to different antibiotics.

[91] Identification of bacteriophages

[92] Five phages against E. coli and five phages against Salmonella enterica were isolated from the sample processing. DNA was then obtained from each of them, and their complete genome was sequenced using commercially available sequencing platforms.

[93] The obtained viral genomes were analyzed in silico to (i) describe the type of viral DNA packaging, (ii) determine the viral DNA replication cycle, (iii) detect the presence of coding sequences for bacterial integrases, virulence factors and resistance genes, and (iv) establish the taxonomic identity of the bacteriophages.

[94] Through in silico analysis of the obtained sequences, a similarity analysis was performed which showed that the obtained genes presented a 6% difference from other reported genes. Thus, the obtained bacteriophages have not been previously reported (Table 4). Bacteriophage ID in Table 4 corresponds to the identification code of the bacteriophage as described in the present disclosure.

Table 4. In silico analysis of viral DNA from isolated bacteriophages.

[95] Example 2: Antibacterial effect of bacteriophages on E. coli ETEC and STEC and on reference strains.

[96] Bacteriophage suspension preparation and viral titer assay on reference strains

[97] Suspensions of the five obtained bacteriophages against E. coli were prepared in TSB media. Each of the bacteriophage suspensions were inoculated separately with a culture or saturated culture of a reference strain of E. coli 0157:H7, such as ATCC 43888. This inoculum was incubated for a period of 18 hours at 37°C with constant shaking at 200 rpm.

[98] To purify the bacteriophages from the culture, the culture was centrifuged at 3200 g for 5 min to separate bacteria and large particles. The supernatant was filtered using a 0.45 pm porosity polyethersulfone (PES) membrane.

[99] To obtain the viral titer, 15 mL of TSB with agar was plated 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 host bacterial culture and 1 mL of TSB and poured onto the previously prepared TSB plate.

[100] Subsequently, serial dilutions of bacteriophage concentrate were prepared in potencies of 10. These dilutions were inoculated on the plate with TSB for 18 hours at 37°C. After the incubation period, the viral microbiological titer was determined by counting lysis plaque forming units (PFU).

[101] The results of these initial assays shown in Table 5 demonstrate that the bacteriophages of the disclosure are able to totally inhibit the growth of the reference strain of E. coli 0157:H7 ATCC 43888, but not the growth of the intestinal microbiota reference strain (ATCC 25922). “+++” corresponds to Clear lysis halo, “++” corresponds to Opaque lysis halo; and corresponds to No observed lytic activity.

[102] In addition, these initial in vitro assays indicate that the bacteriophage with the highest activity on the reference strain O157:H7 would be the phage EcoM-FR7.

Table 5. Plaque lysis assays of bacteriophage isolates on reference strains.

[103] Subsequently, each of the bacteriophages was tested against the E. coli strains identified as ETEC or STEC. For this purpose, representative strains were chosen from among all the isolated strains, and the obtained results for the lysis plate assays are shown in Table 6. “+++” corresponds to Clear lysis halo, “++” corresponds to Opaque lysis halo; “+” corresponds to Activity is present but no halo; and corresponds to No observed lytic activity. Table 6. Plaque lysis assays of isolated bacteriophages on E. coli ETEC and STEC strains.

+++: Clear lysis halo; ++: Opaque lysis halo; +: Activity is present but no halo; No lytic activity observed

[104] As can be seen from this table, most of the bacteriophages showed lytic activity on at least two environmental strains of E. coli that were isolated from dairy samples, and all showed activity on the reference E. coli 0157:H7 strain.

[105] Determination of multiplicity of infection of bacteriophages on E. co// strains. [106] Next, assays were performed to determine the minimum MOI of each of the bacteriophages on E. coli O157:H7 ATCC 43888 and on the ATCC 25922. The obtained results are shown in Table 7.

Table 7. Minimum MOI determined for reference strains and for a STEC strain.

[107] When analyzing these results, it can be concluded that phage PHT-EC-B5 cannot be removed from the bacteriophage cocktail because it is the only phage that showed activity on strain 86A. The same applies to phage EcoM-FR3 since it was the only phage that presented high lytic activity on strain 12B. Finally, phage EcoM-FR5 was the only phage that showed activity on strains 13B and 86B, so it could not be removed from the mix either. On the other hand, bacteriophages EcoM-FR6 and FR7 showed activity against the same E. coli strains against which the other phages showed activity. Further, FR6 bacteriophage had the higher minimal MOI between the assayed, and it is not desirable to have a bacteriophage with higher minimal MOI from a production point view.

[108] Based on the results detailed in Table 6 and 7, the bacteriophages PHT-EC- B5, EcoM-FR3 and EcoM-FR5 were selected, which in combination, allow inhibiting the growth of at least the 3 serovars of interest.

[109] These results demonstrate that it is necessary to carefully determine which bacteriophages can be combined in a cocktail to function as a commercial product.

[110] Example 3: Antibacterial effect of bacteriophages on environmental and reference Salmonella enterica strains.

[111] Evaluation of the inhibitory effect on bacterial growth.

[112] A culture of the bacterium of interest (S. Typhimurium, S. Mbandaka, S. Infantis, S. anatum and S. Panama obtained from repository and environmental isolates) was mixed in TSB medium having OD600=0.3, and poured onto a previously prepared TSB plate. A dilution of each bacteriophage suspensions to an initial MOI greater than 105 PFU/CFU were inoculated on the plate with TSB for 18 hours at 37°C. After the incubation period, the viral microbiological titer was determined by counting lysis plaque forming units (PFU).

[113] In this assay, the identification of 5 bacteriophages with antimicrobial activity against some of the S. enterica strains of interest was achieved.

Table 8. Host range of 5 bacteriophages isolated shows lytic activity on repository and environmental isolates of Salmonella enterica strains.

lytic activity observed

COT038: correspond to environmental isolates

R_003: It is a reference bacteria obtained from the ATCC collection.

[114] As can be seen, the growth of the repository and environmental isolated bacteria is affected by each of these bacteriophages.

[115] When analyzing these results, it can be concluded that phage SenM-L8 cannot be removed from the bacteriophage cocktail because it is the only phage that showed activity on serovar Mbandaka. The same applies to phage SenM-M7 since it was the only phage that presented high lytic activity on serovar Infantis. Finally, phage SenS-STM47B was the only phage that showed activity on serovar Anatum, so it could not be removed from the mix either. Based on the results detailed in Table 8, the bacteriophages SenM-L8, SenM-M7 and SenS- STM47B were selected, which in combination, allow inhibiting the growth of at least the 5 serovars of interest.

[116] These results demonstrate that it is necessary to carefully determine which bacteriophages can be combined in a cocktail to function as a commercial product.

[117] Example 4: Formulation of bacteriophages with lytic activity on Salmonella enterica and E. coll ETEC and STEC isolates.

[118] The formulation comprising the bacteriophages of the disclosure is prepared by the following steps:

B. At the end of the incubation period, the bacteriophages are purified from the culture medium by centrifugation and the use of 0.22 pm microcellulose filters, or other compatible material;

D. At least two of the six individual formulations obtained in the previous steps are mixed: SenM-L8 bacteriophage (IDAC deposit 060820-01), SenM-M7 bacteriophage (IDAC deposit 060820-06), SenS-STM47B bacteriophage (IDAC deposit 060820-05), PHT-EC-B5 bacteriophage (ATCC deposit PTA-122323), EcoM-FR3 bacteriophage (IDAC deposit 121 121 -08), and EcoM-FR5 bacteriophage (IDAC deposit 200520-01 ). In this step, appropriate amounts of each of the individual formulations are added so that each of the bacteriophages is left at a final concentration of between 1 x104 to 1 x1010 PFU/mL;

E. The formulation obtained in step D. is combined with a matrix feed in a ratio between 2-10 of bacteriophages mixtures and between 20 and 70% w/v food matrix;

I. Dry the sample in a suitable industrial equipment until obtaining a solid product with a humidity between 0.001 % and 10%.

[119] The formulation obtained in step I. was evaluated in in vivo assays as is showed in the following example.

[120] Example 5: Evaluation of the bacteriophages efficacy in reducing neonatal diarrhea episodes in calves caused by Salmonella enterica and E. coll ETEC and STEC.

[121] Study 1

[122] The first efficacy trial of the formulation of the disclosure to control episodes of neonatal diarrhea in calves was conducted in a dairy in Brazil. For these trials, newborn calves were fed colostrum and were separated into two groups. The first group consisted of 100 calves and was the negative control group since the animals were given the formulation without bacteriophages. The second group also consisted of 100 animals and was the study group to which the formulation of the disclosure was administered. [123] The parameters measured during the study were the following: (1 ) birth weight, (2) weight at 30 days, and (3) weight at 70 days.

[124] The obtained results show that the duration of the first episode of diarrhea in the control group was significantly shorter compared to the control group (Figure 3). It was also observed that the duration of diarrhea episodes in the first 30 days of life of calves was significantly lower in the study group (which received the formulation of the disclosure) compared to the control group (Figure 4).

[125] In addition, the average daily weight gain in the first 30 days of life was significantly higher in the study group compared to the control group (Figure 5). These results are consistent with those observed for the first 42 days of life (partial weaning), and for the 80 days of life (total weaning) (Figure 6).

[126] Study 2

[127] The second efficacy trial of the formulation of the disclosure to control episodes of neonatal diarrhea in calves was conducted in a dairy in Brazil. For these trials, newborn calves were fed colostrum, and were separated into two groups. The first group consisted of 17 calves and was the negative control group since the animals were given the formulation without bacteriophages. The second group also consisted of 17 animals and was the study group to which the formulation of the disclosure was administered.

[128] The parameters measured during the study were the following: (1 ) birth weight, (2) weight at 30 days, (3) weight at 70 days, and (4) severity of diarrhea. Scoring for assigning diarrhea severity was as follows: 0 point if no diarrhea was visualized, 1 point if moderate diarrhea was observed, and 2 points if severe or bloody diarrhea was observed.

[129] The obtained results show that the severity of diarrhea observed in the animals was significantly lower in the study group compared to the control group (Figure 7).

[130] In relation to the weight gain of the animals during the first week of rearing, it was observed that the study group (to which the formulation of the disclosure was administered) obtained a 10% higher weight gain compared to the control group (Figure 8). These results are consistent with the weight gains observed at 24 days and 70 days of rearing (Figure 9).

[131] While preferred embodiments of the present disclosure 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 disclosure be limited by the specific examples provided within the specification. While the disclosure 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 disclosure. Furthermore, it shall be understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of disclosure and variables. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is therefore contemplated that the disclosure shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. An antibacterial formulation comprising at least two bacteriophages having lytic activity against enterotoxigenic Escherichia coli (ETEC) strains, against Shiga toxin producing Escherichia coli (STEC) strains and against Salmonella enterica, comprising: a) an effective amount of at least two of the following bacteriophages:

Bacteriophage PHT-EC-B5 (ATCC deposit PTA-122323)

Bacteriophage EcoM-FR3 (IDAC deposit 121 121 -08)

Bacteriophage EcoM-FR5 (IDAC deposit 200520-01)

Bacteriophage SenM-L8 (IDAC deposit 060820-01 )

Bacteriophage SenM-STM1 (IDAC deposit 060820-03)

Bacteriophage SenS-STM47B (IDAC deposit 060820-05) b) a food matrix; and c) optionally a chelating salt.

2. The antibacterial formulation of claim 1 , wherein said bacteriophages are at concentrations of 1 x10⁴-1 x10¹° PFU/mL.

3. The antibacterial formulation of claim 1 or 3, wherein said bacteriophages are at concentrations of 1 x10⁶-1 x10⁹ PFU/mL.

4. The antibacterial formulation of claim 1 to 3, wherein said bacteriophages are at concentrations of 1 x10⁷-1 x10⁸ PFU/mL.

5. The antibacterial formulation according to any one of claims 1 to 4, wherein it is formulated as a veterinary formulation for oral administration.

6. The antibacterial formulation according to any one of claims 1 to 5, wherein it is formulated as a veterinary formulation for oral administration in liquid or powder form.

7. The antibacterial formulation according to any one of claims 1 to 6, wherein it is formulated as a veterinary formulation for oral administration in powder form.

8. Use of the antibacterial formulation according to any one of claims 1 to 7 wherein it serves for the prevention and treatment of infectious diseases caused by enterotoxigenic and enterohemorrhagic Escherichia coli, and by Salmonella enterica serovars which are selected from Infantis, Typhimurium, Mbandaka, Anatum, Dublin and Panama wherein the infectious disease is colibacillosis and salmonellosis.

9. Use of claim 8, wherein the STEC and ETEC serotypes are O157:H7 and O25:H10, respectively.

10. The use of claim 8 or 9, wherein it serves to prepare a medicament useful for the prevention and treatment of colibacillosis in a non-human animal such as bovine, ovine or caprine animal.

1 1 . The use of any one of claims 8-10, wherein the non-human animal is a calf.

12. The use of claim 1 1 , wherein the calf is a newborn calf.

13. A method for preventing or treating infectious diseases caused by Escherichia coli ETEC and STEC, and Salmonella enterica, wherein it comprises administering the antibacterial formulation described in claims 1 to 7 to cattle orally.

14. The method of claim 13, wherein it comprises administering is performed during the first 80 days of life.

15. The method of claim 14, wherein it comprises administering is performed daily during the first 80 days of life.

16. The method of claim 12, wherein it comprises administering is performed daily during the first 42 days of life.

17. The method of claim 12, wherein it comprises administering is performed daily during the first 25 days of life.

18. The method of claim 12, wherein it comprises administering is daily during the first 7 days of life.

19. The method of claim 12, wherein it comprises administering is performed daily during the first 3 days of life.

20. A method for producing a microencapsulated solid bacteriophage formulation, comprising the following steps: a. propagating each bacteriophage separately with its respective host bacterium using standard culture conditions; b. at the end of the incubation period, purifying said bacteriophages from the culture medium by centrifugation and use of 0.22 pm microcellulose filters or other compatible material; c. quantifying each bacteriophage formulation in terms of the number of PFU/mL obtained by standard methodologies; d. mixing at least two of the six individual formulations obtained in the previous steps: SenM-L8 bacteriophage (IDAC deposit 060820-01 ), SenM-M7 bacteriophage (IDAC deposit 060820-06), SenS-STM47B bacteriophage (IDAC deposit 060820-05), PHT- EC-B5 bacteriophage (ATCC deposit PTA-122323), EcoM-FR3 bacteriophage (IDAC deposit 121 121 -08), and EcoM-FR5 bacteriophage (IDAC deposit 200520-01 ), wherein appropriate amounts of each of said individual formulations are added so that each of said bacteriophages is left at a final concentration of between 1 x104 to 1 x1010 PFU/mL; e. combining said formulation obtained of step d. with a matrix feed in a ratio between 2-10 of bacteriophages mixtures and between 20 and 70% w/v food matrix; f. adding a chelating agent, or one of its salts, preferably EDTA, in a proportion between 0.1 % and 0.3% w/v in relation to the amount of the mixture obtained in step e.; g. adding water in a proportion of at least between 40 and 80% v/v in relation to the composition obtained in step f.; h. mixing the mixture obtained in step g. for at least 24 hours, until obtaining homogeneity; and i. drying the sample in a suitable industrial equipment until obtaining a solid product with a humidity between 0.001 % and 10%.

20. The method of claim 19, wherein the feed matrix is selected from Molasses, Corn, Soybean, Wheat, Rice, Barley or Rye and any other technical equivalent compatible with formulations acceptable in the veterinary industry.

21 . The method of claim 19 or 20, wherein the bacteriophages of the formulation are selected from: bacteriophage PHT-EC-B5 (ATCC deposit PTA-122323); bacteriophage EcoM-FR3 (IDAC deposit 121 121 -08); bacteriophage EcoM-FR5 (IDAC deposit 200520-01 ); bacteriophage SenM-L8 (IDAC deposit 060820-01); bacteriophage SenM-STM1 (IDAC deposit 060820-03); bacteriophage SenS-STM47B (IDAC deposit 060820-05).