# Patent

## Title
Salmonella bacteriophage compositions and uses thereof

## Abstract

The present invention relates to novel bacteriophages and bacteriophage cocktails containing the novel bacteriophages and or parts and/or products of them, all of them belonging to the Caudovirales order. The novel bacteriophages and the bacteriophage cocktails containing the novel bacteriophages and or parts and/or products of them are Salmonella specific bacteriophages and have lytic activity against Salmonella.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This is a National Stage of International Application No. PCT/EP2012/064797 filed Jul. 27, 2012, claiming priority based on Spanish Patent Application No. 11382257.1 filed Jul. 27, 2011, the contents of all of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention relates to novel bacteriophages and bacteriophage cocktails containing the novel bacteriophages and or parts and/or products of them, all of them belonging to the Caudovirales order. The novel bacteriophages and the bacteriophage cocktails containing the novel bacteriophages and or parts and/or products of them are Salmonella specific bacteriophages and have lytic activity against Salmonella. The novel bacteriophages and the bacteriophage cocktails containing the novel bacteriophages and/or parts and/or products of them, when administered to animals are useful for the control of different Salmonella serovars. The invention refers to the use of the novel bacteriophages and the novel and safe phage cocktail compositions as an antimicrobial for the control of populations of Salmonella in animal therapy and livestock in general, as well as, a sanitization and sterilization composition to be applied on slaughterhouses, animal transportation, food processing industries and foods.
BACKGROUND OF THE INVENTION
Salmonellosis, due to non-typhoid Salmonella enterica serovars, is one of the most frequently reported food-borne diseases worldwide. Since 1993 to 2003, Enteritidis (84%), Typhimurium (7%), Virchow (1%), Infantis (1%) and Blockley (1%), were the serovars of S. enterica mainly isolated from human salmonellosis in European Union. More recently, data from 2006 to 2007 corroborate that Enteritidis and Typhimurium continue to be the most prevalent, although an increase of Typhimurium (16.5%) and a decrease of Enteritidis (64.5%) must be noted (EFSA, 2010).
Poultry and swine are known reservoirs of these zoonotic pathogens. S. Enteritidis is the most frequent cause of human salmonellosis at European Community level. In general, this serovar is also the most frequently isolated from poultry meat and especially in table eggs, whereas it is less commonly found from pigs and cattle and products thereof. The second most prevalent serovar in humans, S. Typhimurium, is the most frequently isolated serovar in pigs, cattle and products thereof and was also among the top three serovars isolated from broilers and table eggs (EFSA, 2010). It is also well documented that in the last decade, the antibiotic resistance is increasing in non-typhoid S. enterica, which means that animals are both pathogen and resistance reservoirs.
Due to the great concern about Salmonella zoonoses, a new Salmonella control program in breeding flocks of Gallus gallus was implemented. This control program aim to meet the Salmonella reduction targets set by the Regulation (EC) No 1003/2005 and covers the following serovars: S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow and S. Hadar. Likewise, a control or monitoring program for Salmonella in pig herds or slaughter pigs have been implemented in different countries with the aim to have more robust data about Salmonella prevalence in swine production. Different strategies and measures to reduce Salmonella have been proposed. In fact, the intensified control of Salmonella in animal populations, particularly in poultry, and a better hygiene throughout the food chain should be the cause of the decrease of the notification rate of Salmonella cases in the European Union. One of these measures has been the use of life or inactivated vaccines to control S. Enteritidis in poultry. However, some authors doubt of its long term effectiveness (Baumler et al., 2000; Rabsch et al., 2000). In this context, it is necessary to open the scope for further research to explore new approaches to existing ones, and new weapons with which to combat these zoonotic pathogens.
Within this aim, the potential use of bacteriophages in human and animal therapy has been extensively reviewed in these lasts years (Barrow and Soothil, 1997; Barrow, 2001; Sulakvelidze et al., 2001; Summers, 2001; Merrill et al., 2003; Matsuzaki et al., 2005; Parisien et al., 2008;). Moreover, its interest as biocontrol agents of zoonotic bacteria in food and animal production and also of others involved in biodeterioration has also arisen and some strategies have been proposed (Barrow and Soothil, 1997; Joerger, 2002; Callaway et al., 2004; Greer, 2005; Hudson et al., 2005). The special characteristics of phages among which are its high specificity and its ability to selfreplicate when infecting, make them especially attractive to prevent food-borne illness, by using them as biocontrol agents in poultry and swine industries and in food production. However, the narrow host-range of bacteriophages is a great restriction to find good candidate phages for bacteriophage therapy against Salmonella enterica because these phages must be able to infect a wide range of strains and serovars of this bacterial species.
There is a consensus about lytic bacteriophages are the best suitable to achieve a significant reduction of bacterial populations due its rapid bacterial killing. Furthermore, safety criteria related to the spread of virulence genes or antibiotic resistance that could be in the phage genome must also be taken into account. Therefore, it is necessary to obtain genomic data demonstrating the impossibility of a lysogenic cycle and the absence of both potential virulence factors and/or antimicrobial resistance genes in the genomes of phage for therapy applications. In addition, it must be noted that the effectiveness of phages in food is likely to vary with each phage, each food matrix, and with the conditions of application including environmental factors (EFSA, 2009).
There are some works testing the effect of different bacteriophages for their inhibitory effect against Salmonella enterica. Thus, it has been reported a reduction of S. Typhimurium in the digestive tract of chickens after oral inoculation of lytic bacteriophages, although high titter of bacteriophages were needed and the inoculation was performed soon after infection to maximized the effect (Berchieri et al., 1991). More recently, much more significant decreases in S. Enteritidis in the cecal contents of broilers orally inoculated with bacteriophages, some of them belonging to the Myoviridae and Siphoviridae family, have been obtained at different times (Fiorentin et al., 2005a; Atterbury et al., 2007; Filho et al., 2007; Borie et al., 2008) and also in S. Typhimurium in experiments that combine the oral inoculation of bacteriophages with competitive exclusion cultures (Toro et al., 2005). In another field of application which included food safety, treatment with bacteriophages also reduced the concentration of S. Enteritidis in poultry products and turkey (Goode et al., 2003; Higgins et al., 2005; Fiorentin et al. 2005b; Bigwood et al., 2008). However, all these works are partial studies because any of them show nor a morphological and genomic characterization of bacteriophages, the phage host range, the efficiency of bacteriophages for reducing Salmonella in poultry over long periods and its efficiency in different matrices and neither some criteria related to safety.
The present invention describes the novel lytic bacteriophages UAB_Phi20 and UAB_Phi78, belonging to the Podoviriade family and UAB_Phi87, belonging to the Myoviridae family, all of them of the Caudovirales order. Those bacteriophages are Salmonella specific bacteriophages that infect different serovars of Salmonella and a high percentage of clonally unrelated strains of the serovars Typhimurium and Enteritidis. Those bacteriophages maintain their infective ability at a wide pH range. The genome of those bacteriophages does not include any known or similar gene to those involved in bacterial virulence.
The present invention also describes bacteriophage cocktail compositions containing at least one of the bacteriophages and/or parts and/or products of the bacteriophages of the invention. Those cocktail compositions can also contain other lytic bacteriophages and/or parts and/or products of them. The novel bacteriophages, when administered to animals, are useful for the bacteriophage therapy over the time of different Salmonella serovars. The present invention refers to the use of these novel bacteriophages and the novel bacteriophage cocktail compositions to reduce the concentration of different Salmonella serovars, providing doses and schedule of administration in animals and in different matrices. The invention refers to the use of the novel bacteriophages and the novel safe phage cocktail compositions as an antimicrobial for the biocontrol of populations of Salmonella in animal therapy and livestock in general as well as a sanitization and sterilization composition to be applied on slaughterhouses, animal transportation, food processing industries and foods.
OBJECTS OF INVENTION
The present invention embrace the novel bacteriophages UAB_Phi20, UAB_Phi78 and UAB_Phi87 belonging to the Caudovirales order. UAB_Phi20 and UAB_Phi78 belong to the Podoviridae family whereas UAB_Phi87 belongs to the Myoviridae family, and all of them are Salmonella specific bacteriophages. The novel bacteriophages and/or parts and or products of them, when administered to animals, are useful for the control of different Salmonella serovars. The present invention also describes bacteriophage cocktail compositions containing at least one of the novel bacteriophages of the invention and/or parts and products of them. These cocktail compositions are administered to animals and are useful for the control of different Salmonella serovars. The present invention refers to the use of the novel bacteriophage cocktail compositions to control different Salmonella serovars, providing methods and schedule of administration, doses and different matrices on/in which Salmonella titres decrease. The invention refers to the use of the novel safe phage cocktails to reduce populations of Salmonella in food, that is, as an antimicrobial for the control of populations of Salmonella in animal therapy and livestock in general, as well as, a sanitization and sterilization composition to be applied on slaughterhouses, animal transportation, food processing industries and foods.
Bacteriophage (Phage): Refers to any virus that infects prokaryotic cells.
Salmonella and Salmonella serovars: As referred by the present invention, Salmonella is a bacterial genus belonging to Enterobacteriaceae family. The Salmonella genus contains three species: Salmonella enterica, Salmonella bongori and Salmonella subterranea. Salmonella enterica comprises six subspecies. The subspecies I includes pathogenic strains either to humans or to other warm-blooded animals. The strains of Salmonella are serologically classified into serovars (serotypes) using the Kauffmann-White scheme. More than 2,540 serovars have been defined. Among them, S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow and S. Hadar are target to be reduced in breeding flocks of Gallus gallus in the European Union. In relation to human salmonellosis in European Union, the most prevalent serovars are Enteritidis and Typhimurium although an increase of Typhimurium and a decrease of Enteritidis is now seen.
Salmonella specific bacteriophage: Refers to any bacteriophage able to infect specifically Salmonella cells.
Cocktail, phage cocktail, cocktail composition, phage cocktail composition: As comprised by the present invention, all of them are equivalent and can be used without distinction. They refer to any formulation containing at least one of the bacteriophages UAB_Phi20, UAB_Phi78, UAB_Phi87 and/or parts and/or products of them, for example bacteriophages enzymes as enzybiotics and tail proteins. In those cocktails, bacteriophages UAB_Phi20, UAB_Phi78, UAB_Phi87 and/or parts and/or products of them can be in similar or different proportion. Those cocktails additionally can also contain other lytic bacteriophages and/or parts and/or products of them. Those lytic bacteriophages are selected between lytic bacteriophages that specifically infect Salmonella, wherein those bacteriophages do not include any known or similar gene to those involved in bacterial virulence. Mainly, those bacteriophages could belong to some of the three families of bacteriophages (Myoviridae, Siphoviridae and Podoviridae) included in the Caudovirales order that were specific for Salmonella. 
These compositions can also contain any of the known carriers of the state of the art, such as carriers adapted for the use according of the present invention, e.g. solvents, including water-based formulations or other technologies such as encapsulation of bacteriophages or parts and products of them.
Pharmaceutically acceptable carrier: Refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. For formulation of the composition into a liquid preparation, a pharmaceutically acceptable carrier which is sterile and biocompatible may be used such as saline, sterile water, Ringer's solution, buffered physiological saline, etc.
Animals: As comprised by the present invention, animal refers to any warm-blooded animal, included humans. In a preferred embodiment, animals are selected by the group comprising Gallus gallus, turkeys and other avian species, pigs and cattle.
Food: Any plant, animal or any substance or material from plants and animals (vegetables, meat, eggs, etc) that provides nutritional support for the life of humans and animals.
Lytic pathway (cycle): A series of steps after bacteriophage infection that lead to bacteriophage replication and the destruction (lysis) of the bacterial host cell.
Lysogenic pathway (cycle): A series of steps that after bacteriophage infection leads to a genetic state (lysogeny) where the bacteriophage genome is replicate as a prophage along with that of the genome of the bacterial host.
Virulence factors: Molecules synthesized by bacterial pathogen cells that participate in the pathogenic process of those bacteria.
Antimicrobial resistance genes: Genes that codify some molecule that allows the growth of bacterial cell in the presence of an antimicrobial agent to which that bacterial cell is usually susceptible.
Host range of bacteriophages: Diversity of bacterial strains that can be infected by a bacteriophage.
Solid matrices: As comprised by the present invention, solid matrices refer to any surface which may be colonized by Salmonella. Those solid matrices would include animal skin, surfaces of farms, slaughterhouses, poultry and swine barns and/or pens, crates used during animal transportation from farms to the food processing industries and other facilities used during the manufacturing process.
Products of bacteriophages: Any product synthesized by the bacteriophage with antimicrobial activity. Those encoded lytic factors are lysozymes, autolysisn or virolysins, also known as enzybiotics. All of them can act by: i) the formation of channels or pores across the bacterial membrane; ii) inhibiting the cell wall biosynthesis; iii) degradation of cellular wall or membrane; iv) depolarization and perforation of the bacterial membrane and v) the nuclease activity of some phage products (Bernhardt et al., 2002; Parisien et al., 2008).
Enzybiotics: Bacterial cell wall hydrolases (BCWH) that lyse bacterial cells from without. BCWH are enzymes that degrade the major component of the cell wall and cause bacteriolysis (Hermoso et al., 2007).
Parts of the bacteriophages: Any single part of the bacteriophage with antimicrobial activity, like the phage tail complexes or tail spike proteins (Parisien et al., 2008).
OBJECTS OF INVENTION
In one embodiment, the present invention refers to a bacteriophage-composition for the prevention of infectious diseases caused by Salmonella comprising at least one of the Salmonella lytic bacteriophages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them.
In another embodiment, the composition contains at least two of the phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them.
In another embodiment the composition comprises phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them.
In another embodiment, the composition also contains other Salmonella lytic bacteriophages and/or products and/or parts of them. Those lytic bacteriophages do not include any known gene or similar gene to those involved in bacterial virulence. In a preferred embodiment, those lytic bacteriophages could belong to the Caudovirales order selected from a collection of lytic bacteriophages that infect Salmonella. 
In a further embodiment, the bacteriophages and/or parts and/or products of them in the compositions are in the same proportion. In another embodiment, bacteriophages and/or parts and/or products of them in the compositions are in different proportion. In a preferred embodiment, the composition comprises phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 in the same proportion. In another embodiment, the composition contains only two of the phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them. In another embodiment, the composition contains only one of the phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of each of them.
In another embodiment, the compositions of the invention additionally includes a pharmaceutically acceptable carrier, solvent or pharmaceutically acceptable excipient.
In a further embodiment, the invention refers to the compositions of the invention for pharmaceutical use.
In another embodiment, the composition is in the form of a suspension, injectable and/or orally administrable dosage forms, sprays or aerosols, etc.
In a another embodiment, the injectable and/or orally administrable dosage forms are selected by the group comprising tablets, troches, lozenges, aqueous or emulsifier suspensions, powder or granules, emulsions, hard or soft capsules, syrups or elixir.
In another embodiment, the composition of the invention is in the form of an animal feed, drinking water, sanitizier or cleaning solution.
In another embodiment, the invention refers to an animal feed comprising the compositions of the invention. In a further embodiment, the invention refers to drinking water comprising the composition of the invention. In further embodiment, the invention refers to a sanitization or cleaning solution, comprising the compositions of the invention.
In a further embodiment, the invention refers to the use of the compositions of the invention to control Salmonella populations in food and solid matrices. In a preferred embodiment, food is selected by the group comprising vegetables, meat and eggs. In another preferred embodiment, solid matrices are selected by the group comprising skin of animals, surfaces of farms, slaughterhouses, poultry and swine barns and/or pens, crates used during animal transportation from farms to the food processing industries or other facilities used during the manufacturing process.
In another embodiment, the invention refers to the use of the compositions of the invention for the manufacture of a medicament for the treatment or prevention of Salmonella infection in an animal.
In another embodiment, the invention refers to the use of the animal feed, drinking water and sanitizer or cleaning solution of the invention for the manufacture of a medicament for the treatment or prevention of Salmonella infection in an animal.
In a further embodiment, the invention refers to the method of treatment or prevention of Salmonella infection consisting of daily continuous treatment of animals with compositions and/or animal feed and/or drinking water of the invention. In a preferred embodiment, the preferred daily doses per animal are selected between 109 to 1012 pfu/day and animal. In a more preferred embodiment the dose is 1010 pfu/day and animal.
In a further embodiment, the treatment consist of spraying animal skin with compositions of the invention. In a preferred embodiment, the preferred bacteriophage concentration in the compositions is selected from the range comprising 109 to 1012 pfu/ml. In a more preferred embodiment, the concentration is 1010 pfu/ml



DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the agarose gel electrophoresis of Eco RI (lanes 3, 6 and 9), EcoRV (lanes 4, 7 and 10) and HindIII (lanes 5, 8 and 11) restriction patterns of DNA of phages UAB_Phi20 (lanes 3 to 5), UAB_Phi 78 (lanes 6 to 8) and UAB_Phi87 (lanes 9 to 11). Lanes 1 and 13 corresponds to Hindi| bacteriophage lambda DNA marker and lanes 2 and 12 to BstEII bacteriophage lambda and Xphi714 DNA markers. This figure shows the DNA digestion pattern of each novel bacteriophage of the present invention (UAB_Phi20. UAB_Phi78 and UAB_Phi87) with three different restriction enzymes (EcoRI, EcoRV and HindIII). It can be observed that each phage presents an own DNA restriction pattern.
FIG. 2 shows the pulsed-field gel electrophoresis (PFGE) of DNA of phages UAB_Phi20 (lane 3), UAB_Phi78 (lane 4) and UAB_Phi87 (lane 5). Lanes 1 and 7 corresponds to lambda PFGE marker and lanes 2 and 6 to low range PFGE marker. This figure shows the genome size of the three Salmonella bacteriophages of the present invention which are around 40 kb for bacteriophages UAB_Phi20 and UAB_Phi78 and around 80 kb for UAB_Phi87, as determined by PFGE technique.
FIG. 3 shows the electron micrographs of the novel bacteriophages UAB_Phi20 (A), UAB_Phi78 (B) and UAB_Phi87 (C). Bacteriophages were applied to electron carbon coated grids and negative stained with 2% uranyl acetate. The preparations were examined with a transmission electron microscope. Electron micrograph images revealed that the novel bacteriophages UAB_Phi20 and UAB_Phi78 belonged to the Podoviridae family while the novel bacteriophage UAB_Phi87 belonged to the Myoviridae family, all of them of the Caudovirales order.
FIG. 4 illustrates the results of the in vitro killing kinetics when infecting S. Typhimurium (Panel A) and S. Enteritidis (Panel B) with UAB_Phi20 (□), UAB_Phi78 (∘), UAB_Phi87 (Δ), UAB_Phi20 and UAB_Phi87 (concentration 1:1) (X) and a phage cocktail (▪). The phage cocktail contains the UAB_Phi20. UAB_Phi78 and UAB_Phi87at a proportion of 1:1:1. Uninfected bacterial cultures (□) and the time of phage infection (□) are also shown. All killing kinetics assays are assayed using relation phage/bacteria of 1. In this experiment, the killing kinetics of the phage cocktail, composed by UAB_Phi20 and UAB_Phi87 or the three novels phages, over the time is better than those obtained with individual phages
FIG. 5 shows the efficacy of the phage cocktail in reducing Salmonella counts in the cecum of the chicks. The cocktail herein used entails the novel phages of the present invention (UAB_Phi20, UAB_Phi78 and UAB_Phi87) at proportion of 1:1:1. In this assay, chicks were infected with S. Typhimurium by the seeder bird infection method. The phage cocktail was orally administrated to chicks when the digestive tract of chicks was fully colonized by Salmonella (values in the cecum were of 108 cfu/g). The administration schedule of phage cocktail at a dose of 1010 pfu/animal was twice per day on days 4th and 5th after Salmonella infection. Salmonella counts from the cecum of individual phage-untreated chicks (♦) and phage-treated chicks (□) are presented as log10 cfu/g cecum content. Only, a decrease of Salmonella count can be observed during the two first days post-treatment. These data indicate that the treatment with phage cocktail have not a significant effect in reducing the concentration of Salmonella over the time when the digestive system of animals is fully colonized by this bacterium.
FIG. 6 illustrates the efficacy of the phage cocktail in reducing Salmonella counts in the cecum of the chicks. The cocktail herein used entails the novel phages of the present invention (UAB_Phi20, UAB_Phi78 and UAB_Phi87) at proportion of 1:1:1. In these experiments, each chick was orally infected with S. Typhimurium at a final dose of 105 cfu/animal. The phage cocktail was administrated one day before (Panel A) and simultaneously (Panel B) to Salmonella infection. In both cases, the phage cocktail was further re-administrated twice per day on days 1, 2 and 3 post-infection and once a day on days 6, 8, 10, 13, 15 and 17 post-infection. In each administration, the dose of the phage cocktail applied was 1010 pfu/animal. Salmonella counts from the cecum of individual control chicks (♦) and phage-treated chicks (□) are presented as log10 cfu/g cecum content. A high reduction of the Salmonella concentration was seen during the first days post-treatment, indicating a high efficacy of phage cocktail in the first stages of Salmonella colonization. Furthermore, a significant reduction of Salmonella concentration over the time was observed with the administration schedule assayed.
FIG. 7 shows the different serovars and strains of S. enterica used to isolate and to determine the host range of the Salmonella phages, including the novel bacteriophages and the bacteriophages of the phage cocktail herein described. In this table, the year of isolation of these strains and its origin are also included.
FIG. 8 shows the host range of the novel bacteriophages and the bacteriophages of the cocktail of the present invention onto the different serovars and strains that have been used to this purpose. As it can be observed all the serovars assayed are infected by the novel bacteriophages as well as the major part of strains of S. Typhimurium and S. Enteritidis.
FIG. 9 indicates the percentage of infectivity over the time of the novel bacteriophages (UAB_Phi20. UAB_Phi78 and UAB_Phi87) after exposure to pH values ranging from 2.0 to 9.0, simulating different environmental conditions. The infectivity of the novel bacteriophages herein described is only altered at extremely acidic pH values.
FIG. 10 shows the stability of the three novel bacteriophages at 4° C. in MgSO4 10 mM over one year period. This stability is measured as the percentage of infectivity of each phage maintained during this period of time at 4° C. These data indicate a high stability of the novel bacteriophages over long time periods.
FIG. 11 shows the stability of each novel bacteriophage in drinking water at room temperature and at 37° C. over one week. Again, stability is measured as the percentage of infectivity of each phage. This data is of interest for applications of phage cocktail in which the preferred carrier of novel bacteriophages was drinking water.
FIG. 12 illustrates the effect of a phage cocktail treatment for 3 and 6 hours at 32-33° C. in decreasing the Salmonella concentration in swine skin sections. The cocktail herein used was composed by the UAB_Phi20. UAB_Phi78 and UAB_Phi87 phages at a proportion of 1:1:1. A significant reduction of Salmonella concentration over the time was observed, indicating the possibility of application of this novel phage cocktail to decrease the count of Salmonella in swine skin.
FIG. 13 illustrates the effect of the treatment with a phage cocktail for 60 min at room temperature of romaine lettuce pieces, experimentally contaminated whit S. Typhimurium and S. Enteriditis. The cocktail was composed by the UAB_Phi20, UAB_Phi78 and UAB_Phi87 phages at a proportion of 1:1:1. A significant reduction of Salmonella concentration over the time was observed, indicating the application of a phage cocktail to decrease the count of Salmonella in food or in food manufacturing processes.
FIG. 14 shows the effect of the treatment with the novel phages UAB_Phi20, UAB—78 and UAB_Phi87, alone or as a cocktail, for 60 min at room temperature of romaine lettuce pieces, experimentally contaminated whit S. Typhimurium and S. Enteritidis. Data obtained show that individual phages as UAB_Phi20 can be used for the Salmonella control.
FIG. 15 illustrates the effect of a phage cocktail treatment during very short time periods (approximately 5 minutes) in decreasing the Salmonella concentration in chicken breast pieces, conserved during one week at 4° C. after the treatment. The cocktail herein used was composed by the UAB_Phi20, UAB_Phi78 and UAB_Phi87 phages at a proportion of 1:1:1. A significant reduction of Salmonella concentration over the time was observed, indicating the application of this novel phage cocktail to decrease the count of Salmonella in food or in food manufacturing processes at 4° C.



DETAILED DESCRIPTION OF THE INVENTION
The present invention refers to the novel bacteriophages UAB_Phi20, UAB_Phi78 and UAB_Phi87, deposited in GenBank under accession numbers GQ422450 (corresponding to SEQ ID NO: 1), GU595417 (corresponding to SEQ ID NO:2), belonging to the Podoviridae family and JN225449 (corresponding to SEQ ID NO:3), belonging to the Myoviridae family, respectively. Those bacteriophages are Salmonella specific bacteriophages from the Caudovirales order. The novel bacteriophages, when administered to animals, are useful for the biocontrol of different Salmonella serovars. The present invention also describes bacteriophage cocktails containing at least one of the bacteriophages and/or parts and/or products of the bacteriophages of the invention. Those cocktail compositions can also contain other lytic bacteriophages and/or parts and/or products of them. In those cocktails, bacteriophages and/or parts and/or products of them can be in similar or different proportion. Those lytic bacteriophages are selected between lytic bacteriophages that specifically infect Salmonella, wherein those bacteriophages do not include any known gene or similar gene to those involved in bacterial virulence. The present invention comprises the use of the novel bacteriophages and the novel bacteriophage cocktail compositions to control different Salmonella serovars, providing administration schedules and doses and different matrices on/in which Salmonella concentration decreases. The invention refers to the use of the novel bacteriophages and the novel safe phage cocktails as an antimicrobial for the biocontrol of populations of Salmonella in food animal therapy and livestock in general as well as a sanitization and sterilization composition to be applied on slaughterhouses, animal transportation, food processing industries and foods.
Characterization of the Phages UAB_Phi20, UAB_Phi78, UAB_Phi87
In this invention, Salmonella specific bacteriophages were isolated from cloacae or rectal swabs from broilers and pigs. Phages, UAB_Phi20, UAB_Phi78, and UAB_Phi87 were selected in the present study because they were able to infect the 94% of the clonally unrelated strains of S. Typhimurium and S. Enteritidis (67 of the 71 strains assayed) (FIG. 8, Example 3). In addition, phages selected infect the serovars Hadar, Infantis and Virchow (FIG. 8, Example 3).
DNA restriction patterns of the three phages were absolutely different among them (FIG. 1, Example 6).
Electron micrographs of negatively stained phages UAB_Phi20 and UAB_Phi78 revealed icosahedral heads and non-contractile short tails, indicating that phages selected belong to the Podoviridae family of the Caudovirales order. In the same way, the electron micrographs of UAB_Phi87 revealed also icosahedral heads with long, rigid and contractile tail, particular characteristics of the Myoviridae family of the Caudovirales order (FIG. 3, Example 5).
The genome size of phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 is 44.05, 43.94 kb and 87.60 kb, respectively, based on PFGE of the genomic DNA of the three selected phages (FIG. 2, Example 6) and on DNA sequencing data (Example 6). The complete genome sequences of the three phages were deposited in GenBank under accession numbers GQ422450, GU595417 and JN225449, respectively. DNA sequences analysis indicated that no virulence genes are encoded in the genomes of the three phages selected.
Phage Stability to Different Environmental Conditions
The phages selected were sensitive to extreme acidic pH value (pH=2.0) (FIG. 9, Example 4). However, the fact they maintained a survival rate ranging between 60-80% A during 30 min of treatment at pH 2.0, implies their capacity to resist the pass throw the stomach, where the lowest pH value is reached. This data clearly indicated that phages selected can be orally administrated to animals.
The selected phages can be easily maintained at 4° C., because the percentage of infection was more than 95% over one year at that temperature (FIG. 10, Example 4). In addition, the phages selected are able to maintain their infective ability in drinking water at room temperature and at 37° C. during at least 7 days (FIG. 11, Example 4).
Phage Cocktail Compositions
As previously commented, lytic bacteriophages are the best suitable way to achieve a significant reduction of bacterial populations due its rapid bacterial killing, but it is not easy to find good candidates. In any bacterial population, resistant cells to phage infection can easily arise, thus it is expected to get better results if combination of different phages is employed. Taking this into account, the invention proposes cocktails to the biocontrol of different Salmonella serovars. The cocktails of the present invention comprise at least one of the bacteriophages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them. Those cocktail compositions can also contain other lytic bacteriophages and/or parts and/or products of them. In those cocktails, bacteriophages and/or parts and/or products of them can be in similar or different proportion. These lytic bacteriophages are selected from lytic bacteriophages that specifically infect Salmonella, wherein those bacteriophages do not include any known gene or similar gene to those involved in bacterial virulence.
In one embodiment of the present invention, the cocktail comprises phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 in the same proportion. In another embodiment, the cocktail comprises parts of UAB _Phi20, UAB_Phi78 and UAB_Phi87 in the same proportion. In another embodiment, the cocktail comprises products of UAB_Phi20, UAB_Phi78 and UAB_Phi87 in the same proportion. In another embodiment, the cocktail comprises phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and parts and products of them, all in the same proportion. In another embodiment, the cocktail comprises phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and parts of them, all in the same proportion. In another embodiment, the cocktail comprises phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and products of them, all in the same proportion. In another embodiment, the cocktail comprises parts of phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and products of them, all in the same proportion.
In another embodiment, the proportion of phage and/or parts and/or products of UAB_Phi20 in the cocktail is superior to the proportion of phages and/or parts and/or products of UAB_Phi78 and UAB_Phi87. In another embodiment, the proportion of phage and/or parts and/or products of UAB_Phi78 in the cocktail is superior to the proportion of phages and/or parts and/or products of UAB_Phi20 and UAB_Phi87. In another embodiment, the proportion of phage and/or parts and/or products of UAB_Phi87 in the cocktail is superior to the proportion of phages and/or parts and/or products of UAB_Phi20 and UAB_Phi78.
The invention also refers to cocktails which contain only two of the phages UAB_Phi20 UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them. This way, in one embodiment, the cocktail comprises phage UAB_Phi20 and/or parts and/or products of it and phage UAB_Phi78 and/or parts and/or products of it. In another embodiment the cocktail comprises phage UAB_Phi20 and/or parts and/or products of it and phage UAB_Phi87 and/or parts and/or products of it. In another embodiment, the cocktail comprises phage UAB_Phi78 and/or parts and/or products of it and phage UAB_Phi87 and/or parts and/or products of it.
The invention also refers to cocktail which contain only one of the phages UAB_Phi20 UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them. This way, in one embodiment, the cocktail comprises UAB_Phi20 and/or parts and/or products of it; in another embodiment the cocktail comprises UAB_Phi78 and/or parts and/or products of it; and in a further embodiment the cocktail comprises UAB_Phi87 and/or parts and/or products of it.
As comprised by the invention, any of the previously described cocktails (cocktails that contain the three phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them, cocktails that contain only two of the phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them, and cocktails that contain only one of the phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts and/or products of them) can also contain other lytic bacteriophages and/or parts and/or products of them. In those cocktails, bacteriophages and/or parts and/or products of them can be in similar or different proportion. Those lytic bacteriophages are selected from lytic bacteriophages that specifically infect Salmonella, wherein those bacteriophages do not include any known gene or similar gene to those involved in bacterial virulence.
The present invention comprises the use of the novel bacteriophages and the novel bacteriophage cocktail compositions to control different Salmonella serovars, providing administration schedules and doses and different matrices on/in which Salmonella concentration decrease.
In accordance with one aspect of the present invention, the bacteriophage and bacteriophage compositions may be administered into animals in a pharmaceutical formulation or as a component of the animal feed or in their drinking water.
Further, the compositions of the present invention may be administered in a typical manner via any route such as oral or parenteral routes, in particular, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, and inhalation routes.
The compositions of the present invention may additionally include a pharmaceutically acceptable carrier and can be formulated together with a carrier to provide foods, medicines and feed additives.
A pharmaceutically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
For formulation of the composition into a liquid preparation, a pharmaceutically acceptable carrier which is sterile and biocompatible may be used such as saline, sterile water, Ringer's solution, buffered physiological saline, etc.
Examples of oral dosage forms suitable for the composition of the present invention include tablets, troches, lozenges, aqueous or emulsifier suspensions, powder or granules, emulsions, hard or soft capsules, syrups or elixir.
For non-oral administration, the composition of the present invention may be formulated into injections for subcutaneous, intravenous, or intramuscular routes, or sprays inhaled via the respiratory tract, such as aerosols.
As can be deduced by the results of the present invention, the preferred administration schedule of the present invention for the treatment of animals consists on the continuous treatment of these animals with the novel bacteriophages and the novel bacteriophage cocktails to control different Salmonella serovars. This continuous treatment can be achieved by the administration of the novel bacteriophages and/or the novel bacteriophage cocktails by the daily drinking water and or daily feed.
In this way, in accordance with another aspect, the present invention relates to an animal feed and drinking water, comprising the bacteriophages as an active ingredient. The bacteriophage or composition of bacteriophages of the present invention may be separately prepared as a feed additive, and then added to the animal feed, or directly added to the animal feed.
The invention refers to the use of the novel bacteriophages and the novel safe phage cocktails as an antimicrobial for the biocontrol of populations of Salmonella in food animal therapy and livestock in general, as well as, a sterilization composition to be applied on a surface of a target in food processing industries and foods.
In this way, in accordance with still another aspect, the present invention relates to a sanitization and a cleaning solution, comprising the bacteriophages or composition of bacteriophages of the present invention as an active ingredient which can be used in solid matrices as surfaces of farms, slaughterhouses, poultry and swine barns and/or pens, crates used during animal transportation from farms to the food processing industries or other facilities employed during the manufacturing process.
In Vitro Killing Assays
To know the killing kinetics of Salmonella infected with the phages of the invention in vitro killing assays were performed, using S. Enteritidis and S. Typhimurium. As previously said, these bacteriophages UAB_Phi20 (accession number GQ422450), UAB_Phi78 (accession number GU595417) and UAB_Phi87 (accession number JN225449) were identified in previous assays carried out in the present invention. They belong to the Caudovirales order, and infect S. Typhimurium and S. Enteritidis. The genome of all phages does not encode any virulence gene and are stable to different environmental conditions. These characteristics make those bacteriophages useful for the purposes of the invention.
The killing kinetics of Salmonella infected with the phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 followed a similar pattern for both serovars studied (FIG. 4, Example 7). As expected, the combined action of a phage cocktail, composed by UAB_Phi20, and UAB_Phi87 (1:1) and UAB_Phi20, UAB_Phi78 and UAB_Phi87 (1:1:1) in decreasing the Salmonella concentration was faster and prolonged along the time than the effect of the treatment of bacterial cultures with each one of the individual phages (FIG. 4, Example 7).
Thus, a decrease of the S. Typhimurium concentration of 3 log10 cfu/ml was reached after 30 minutes of addition of a phage cocktail composed by the three novel phages. This level of bacterial reduction was maintained during 8 hours, with a maximum of 4.4 log10 cfu/ml (FIG. 4 panel A and Example 7). Both values of reduction for S. Enteritidis treated with the phage cocktail were 4 log10 cfu/ml and 6.5 log10 cfu/ml, respectively (FIG. 4 panel B and Example 7). Regarding these killing kinetics the effect of a phage cocktail reported here on Salmonella is better than that described by other phage cocktail after 4 hours of phage infection (Toro et al., 2005).
Those results make the phages UAB _Phi20, UAB_Phi78 and UAB_Phi87 appropriated for the purposes of the invention.
In Vivo Killing Assays, Administration Schedules and Preferred Doses
After the in vitro killing assays, in vivo killing assays were made, that is, the effect of a phage cocktail on S. Typhimurium concentration in a chicken model (White Leghorn chicken specific pathogen free). In all assays, the phage cocktail was composed by UAB_Phi20, UAB_Phi78 and UAB_Phi87 (1:1:1).
Previously, the residence time of phages in the chick digestive system was determined by oral administration of the phage cocktail at a dose of 1010 pfu of each phage/animal. The results of the present invention show that the residence time of phages in the intestinal tract ranged from 7 to 9 days, but a significant decrease in the phage concentration was detected from the 4 day of phage administration to animals.
Two types of S. Typhimurium infection of chicks were experimented in in vivo killing assays. In one of them, Salmonella infection was performed by the seeder bird infection method. Whit this procedure, the Salmonella cecum content of chicks was 108 cfu/g, approximately. In the second one, chickens were experimentally infected by oral inoculation with Salmonella at a dose of 105 cfu/animal. In both cases, the effect of phages was assessed by measuring the decrease in the Salmonella concentration in the cecum of animals. The concentration of each phage in the cocktail was 1011 pfu/ml and 0.1 ml of that cocktail were orally administrated to animals.
The administration of four doses of phage cocktail to chicken fully colonized by Salmonella did not show a significant reduction of the Salmonella concentration in the cecum of animals (FIG. 5, Example 8). In contrast, chicks experimentally infected with 105 Salmonella cells per animal and treated with the phage cocktail, showed that the best efficacy of phage cocktail was achieved when the administration schedule of phages was one day before (FIG. 6 panel A, Example 8) or just (FIG. 6 panel B, Example 8) to bacterial infection and successively re-administrated different days after the first doses. In these cases, the administration schedule of phages in reducing S. Typhimurium was significantly (p<0.0001). In both experiments, a reduction of 4 log10 cfu/g on the cecal content was reached during the first days post-treatment and a reduction of 2 log10 cfu/g was achieved at the end of the experiment (FIG. 6, Example 8). With this strategy the present invention achieves better results in Salmonella reduction over time than those reported by other authors (Toro et al., 2005; Atterbury et al., 2007; Borie et al., 2008; Filho et al, 2007). This result probably can be generalized to the application of phages to decrease undesired bacterial populations in the intestinal tract of animals
As can be deduced by the results of the present invention, the preferred administration schedule of the present invention consist on the continuous treatment of animals with the novel bacteriophages and the novel bacteriophage cocktails to control different Salmonella serovars.
Taking all those results into account, the preferred embodiment of the present invention refers to the daily continuous treatment of animals with phages and/or cocktails of the invention. In one embodiment of the present invention, the preferred daily doses per animal may vary between 109 to 1012, preferably 1019 pfu/day and animal.
This continuous treatment can be achieved by the administration of the novel bacteriophages and/or the novel bacteriophage cocktails by the dairy drinking water and/or feed.
Application of the Phage Cocktail to Reduce Salmonella in Slaughterhouses
As previously said, the invention refers to the use of the novel bacteriophages and the novel safe phage cocktails as an antimicrobial for the biocontrol of populations of Salmonella in animal therapy and livestock in general as well as a sanitization composition to be applied in slaughterhouses, animal transportation, food processing industries and foods. This comprises a sanitization and a cleaning solution, comprising the bacteriophages or composition of bacteriophages of the present invention as an active ingredient which can be used in the surfaces of farms, slaughterhouses, poultry and swine barns and/or pens, crates used during animal transportation, contaminated areas and other production facilities.
The pre-slaughter period is considered crucial for the microbial quality of the final products in the food production chain (Lo Fo Wong et al., 2002). In this way, animal skin with absence or minimal bacterial colonisation is essential for a safety food available for human consumers. In this sense, at our knowledge, there is not any report about the application of phages to reduce the Salmonella concentration in swine skin.
In this invention, swine skin samples (1500 cm2) acquired in a butchery were experimentally contaminated with either S. Typhimurium or S. Enteritidis at a concentration of 102 cfu/cm2. Afterwards, skin pieces were sprayed with a solution containing the phage cocktail (1010 pfu of each phage/ml), dried and maintained 6 hours at 33° C. This temperature condition simulates the skin temperature of pigs (Huynh et al., 2005) and the ideal lairage time (Warriss et al., 1998). In both cases, Salmonella reduction was extremely strong (p<0.005). In fact, after 3 h of treatment, direct colony count was not possible in both serovars (FIG. 12, Example 9).
The present invention is the first report showing reduction of Salmonella in swine skin mediated by a phage cocktail. For this reason, the results obtained cannot be compared with other studies. Overall, this invention could be used for reducing Salmonella on animal skin in the slaughterhouse during the withdrawal time.
Taking these results into account, a preferred embodiment of the present invention refers to spray animal skins with a solution of the novel bacteriophages and/or the novel bacteriophage cocktails in drinking water or in any other carrier for 3 hours at room temperature before slaughter (FIG. 12, Example 9). In one embodiment of the present invention, the preferred bacteriophage concentration may vary between 108 to 1012 pfu/ml, more preferred 1010 pfu/ml.
Application of the Phages and Phage Cocktail to Reduce Salmonella on Food Processing Industries and Foods
A relevant challenge for food production is to assure food safety by coordinated control measures applied in different steps of the food chain (from Farm to Fork). Foods are a source of Salmonella, and not only products derived from animals but also vegetables. It is well known the presence and the persistence of Salmonella in many vegetables, including lettuce (Wells and Butterfield, 1997; Kakiomenou et al., 1998; Weissinger et al., 2000; Natvig et al., 2002; Islam et al., 2004). In this way, another object of the present invention is the application of the novel phages and/or cocktails of the invention on foods to reduce Salmonella. 
In this invention, lettuce samples were experimentally infected with either S. Typhimurium or S. Enteritidis at a concentration comprised between 104 and 105 cfu/g. Afterwards, lettuce samples were submerged into a solution containing the phage cocktail (109 pfu of each phage/ml). The phage cocktail was composed by UAB_Phi20, UAB_Phi78 and UAB_Phi87 (1:1:1). The highest effect of phage cocktail was found in reducing the S. Typhimurium concentration (FIG. 13, Example 10). A 3.4 log10 cfu/g and 1.9 log10 cfu/g reduction was reached in lettuce after 30 min of treatment for S. Typhimurium and S. Enteritidis, respectively (p<0.0001) (FIG. 13, Example 10). Phage UAB_Phi20 was the most effective to reduce Salmonella, reaching to similar values as the obtained with the bacteriophage cocktail after 60 min of treatment of lettuces experimentally infected with S. Typhimurium and S. Enteritidis (FIG. 14, Example 10).
The present invention is the first report showing a significant reduction of Salmonella in vegetables mediated by bacteriophages and a phage cocktail.
Furthermore, similar to what occurs with lettuce, the phage cocktail of the present invention produces a reduction of the number Salmonella in chicken breast pieces experimentally infected with 104 cfu/g of Salmonella. In this case, reductions of 3.9 log10 and 1 log10 cfu/g of S. Typhimurium and S. Enteritidis, respectively (p<0.0001), were obtained after the conservation in the refrigerator (4° C.) during 7 days of chicken breast pieces, treated for 5 min with the bacteriophage cocktail (FIG. 15, Example 10). Other studies of treatment of chicken meat with phages have been reported. However, their experimental designs were different that those here performed (Goode et al., 2003; Fiorentin et al. 2005b; Higgins et al., 2005; Bigwood et al., 2007). Therefore, a direct comparison among our experiments and those reported would not be adequate.
Taking all those results into account, a preferred embodiment of the present invention refers to the treatment of vegetables with phages and/or cocktails of the invention. In one embodiment of the present invention, the preferred way of treatment is to maintain the food submerged for 30-60 min at room temperature into a solution of the novel bacteriophages and/or the novel bacteriophage cocktails in drinking water or in any other carrier. In one embodiment of the present invention, the preferred bacteriophage concentration may vary between 108 to 1012 pfu/ml, more preferred 109 pfu/ml.
In another embodiment of the present invention, a preferred way of treatment of food is to submerge them for 5-10 min at 4° C. into a solution of the novel bacteriophages and/or cocktails in saline solution or in drinking water (FIG. 11, Example 4). In one embodiment of the present invention, the preferred bacteriophage concentration may vary between 108 to 1012 pfu/ml, more preferred 109 pfu/ml.
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EXAMPLES
The invention is now described with reference to the following examples. These examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these examples but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.
Statistical Analysis
Non parametric statistics (Mann-Whitney-Wilcoxon test) was used to determine the significance of phage cocktail administration in the different chicken trials. Parametric statistics (Generalized linear model) was used to determine the significance of phage and phage cocktail treatments of food. The significance level was fixed at 5%.
Example 1
Bacterial Strains and Growth Conditions
Clonally unrelated strains of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, respectively, were used to isolate Salmonella's phages and to determine the host range assays of the isolated phages (FIG. 7). These bacterial strains were obtained from the Laboratori de Sanitat Animal (DARP, Generalitat de Catalunya, Spain) and from the Hospital Vall d'Hebrón and the Hospital de Sant Pau (Barcelona, Spain). They were selected to present different pulsotypes as determined by Pulse Field Gel Electrophoresis method.
The virulent strains S. Typhimurium ATCC14028 (American Type Culture Collection) and S. Enteritidis LK5 (Salmonella Genetic Stock Centre, University of Calgary) were also used for the isolation of Salmonella's phages. In addition and using the red recombinase system (Datsenko and Wanner, 2000), the ATCC14028 and LK5 virulent strains were respectively marked with a chloramphenicol and kanamycin resistance cassette in an intergenic sequence, generating ATCC14028 ΩCm and LK5 ΩKm strains. These strains were employed for efficacy studies of the selected bacteriophages.
Moreover, the strains 05S72, 10152 and 8546 of S. Hadar, the strains 05S44 and 1056 of S. Infantis and the strains 791/S and 9781 of S. Virchow, were used for the host range studies of the bacteriophages isolated (FIGS. 7 and 8).
All bacterial strains were routinely grown on Luria Bertrani (LB) broth or agar plates, and, when necessary, chloramphenicol (34 μg/ml) and kanamycin (150 μg/ml) were added. Growth was performed at 37° C. for both broth and plate cultures during 18 h.
Example 2
Isolation and Propagation of Bacteriophages
Samples (n=161) of cloacae or rectal swabs from broilers and from pigs, respectively, were taken from farms of different geographical areas of Spain from 2007 to 2009.
Bacteriophages were isolated as described (Connerton et al., 2004; Muniesa et al., 2005) or by an enrichment procedure. Briefly, 1 g of the sample was diluted in 10 ml of SM buffer (50 mM Tris-HCl [pH 7.5], 0.1 M NaCl, 8 mM MgSO4·7 H2O, 0.01% gelatin) and incubated at 4° C. for 24 h. Then each suspension was subjected to centrifugation at 7000×g for 10 min. For the enrichment procedure 1 g of the sample was diluted in 10 ml of peptone water and incubated at 37° C. for 18 h. After then, 1 ml of the enrichment culture was inoculated in 10 ml of Müller-Kauffmann selective broth and incubated at 37° C. for 24h. Following, each culture was subjected to centrifugation at 7000×g for 10 min.
The supernatants obtained from both methods were filtered through a 0.45 μm syringe filter. Then 10 μl of each filtrate was spotted onto the surfaces of Salmonella lawns by the double agar layered method. After incubation at 37° C., phage plaques were visualized and each different plaque was pushed and resuspended in 1 ml of sterile MgSO4 10 mM. Afterwards, 5 dilutions (1:10) were made and plated using the double agar layered using the same Salmonella tester strain. This procedure was repeated at least three subsequent times to purify the bacteriophages.
Bacteriohage lysates were obtained by infecting different Salmonella strains during 5 h at 37° C., grown in LB broth. After centrifugation of cultures at 7000×rpm for 10 min, the supernatant was filtered through a 0.45 μm syringe filter and the phage titter was determined by plating serial dilutions onto LB plates using the double agar layered method. When necessary, bacteriophages were purified by ultracentrifugation at 51.000×g for 2 h.
Example 3
Selection of Bacteriophages
The host range of each bacteriophage lysate was determined by spotting 10 μl of the lysate (108 pfu/ml) onto lawns of 76 strains of Salmonella. Plates were incubated at 37° C. for 24 h and bacterial lysis was recorded. Bacteriophages selected infect to the serovars Hadar, Infantis and Virchow and a wide range of strains belonging to the serovars Typhimurium and Enteritidis (FIG. 8).
In addition, DNA restriction profiles of the phage selected were also determined. To achieve this, a purified phage suspension was treated with DNase I (20 μg/ml) and RNase I (25 μg/ml) at 37° C. for 1 h. Hereafter, proteinase K (0.2 mg/ml) and sodium dodecyl sulfate (0.5%) were added and incubated for 1 h at 56° C. Following incubation, the DNA was purified by conventional phenol-chloroform-isoamyl alcohol extraction. The genomic DNA was precipitated in 2 volumes of ethanol with 3 M sodium acetate, pH 4.8. The pellet obtained was washed with 70% ethanol, dried, and resuspended in 100 μl MQ grade water. Phage DNA was digested with restriction endonucleases EcoRI, EcoRV and HindIII (Roche) according to the supplier's recommendations. DNA fragments were subjected to electrophoresis on agarose gel (0.7% and containing ethidium bromide) in Tris-acetate-EDTA (TAE) buffer at constant voltage of 80 V and visualized by UV light. DNA standard fragments of HindIII and BstEII bacteriophage lambda were used as markers to calculate the size of DNA fragments.
Phages UAB_Phi20, UAB_Phi87, isolated from cloacae broiler swabs, and UAB_Phi78 from pigs were selected for further characterization regarding their host range and their DNA digestion pattern (FIG. 1).
Example 4
Phage Stability to Different Environmental Conditions
The effect of pH on infectivity of phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 of the present invention was determined at 25° C. in MgSO4 10 mM at pH ranging from 2.0 to 9.0. Phage samples were taken each 30 min for 2 h and were immediately serially diluted and plated using the double agar layered method. Plates were incubated at 37° C. for 24 h and phage plaques counted.
Phage stability in MgSO4 10 mM was recorded during one year at 4° C. In addition, phage stability in drinking water was also recorded during one weak at room temperature and at 37° C. Lysates titration was performed as previously described.
The effect of pH, ranging from 2.0 to 9.0 on the infectivity of the three phages was determined (FIG. 9). All three phages maintained a percentage of infectivity greater than 90% for 120 min at pH values of 4.0, 6.0 and 9.0, except UAB_Phi87 and UAB_Phi78 phages which have a percentage of infectivity of 85.2% and 87.8%, respectively, at pH 4 to 120 min. In contrast, the stability of all the three phages dramatically decreased at pH 2.0. The most sensitive to acidity was UAB_Phi87, while UAB_Phi20 was the most stable phage.
The stability of the three phages maintained during one year at 4° C. was also determined. The percentage of infection was more than 95% over the period studied (FIG. 10). Only, UAB_Phi78 showed a slightly reduction regarding the other phages. Likewise, all the three phages presented a very good stability in drinking water during 7 days (FIG. 11). No alteration of the infectivity was appreciated over this period of time at room temperature. However, slightly reductions were observed on the infectivity of the three novel bacteriophages herein described at 37° C. Nevertheless, the infectivity of them was greater than 90% after one week under this condition.
Those results demonstrated that phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 of the invention can be easily maintained in some carrier as MgSO4 10 mM during a long period of time at 4° C. Furthermore, these carriers and drinking water can be used as vehicle for administration purpose of the invention. The high stability of phages to low pH values demonstrated that phages of the invention UAB_Phi20, UAB_Phi78 and UAB_Phi87, can be orally administrated and then used for the purpose of the invention.
Example 5
Electron Microscopy
Purified phages (109-1010 pfu/ml) were applied to electron carbon coated grids and negative stained with 2% uranyl acetate. After drying, preparations were examined with a JEOL 1400 transmission electron microscope at different magnitudes. Phage morphology and dimensions were recorded.
UAB_Phi20 and UAB_Phi78 phages morphologically belong to the Podoviridae family of the Caudovirales order, while UAB_Phi87 belong to the Myoviridae family of the same order (Ackermann, 1998). Thus, electron micrographs of negatively stained phage UAB_Phi20, UAB_Phi78 revealed icosahedral heads and non-contractile short tails while the electron micrograph of UAB_Phi87 revealed also icosahedral head but a long, rigid contractile tail (FIG. 3). The head dimensions of UAB_Phi20,UAB_Phi78 and UAB_Phi87 were 60±1.5 nm, 66±1.7 nm and 68±2.7 nm, respectively and tail length was of 13±0.7 nm, 14±0.7 nm and 114±4.3 nm, respectively.
Example 6
Genome Characterization 
Phage genome size determination using pulsed-field gel electrophoresis (PFGE) was performed as previously described (Atterbury et al., 2003). Gels were run using a Bio-Rad CHEF DRIII system in 0.5 TBE (Tris-borate 50 mM, EDTA 0.1 mM) for 15 h at 6V/cm with switch time of 2 to 20s during 10 h and 20 to 30 s during 5 h. Gels were stained with ethidium bromide and visualized using the GelDoc System (BioRad). Genome size was determined with Fingerprinting II program (BioRad).
DNA sequencing was done by Sistemas GenOmicos (Spain) using a shotgun strategy. Open reading frames (ORFs) were searched using the NCBI ORF Finder program (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Potential ORFs were compared against the NCBI protein databases using the BLASTP nonredundant database (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Based on PFGE of the genomic DNA of the selected phages (FIG. 2) and on DNA sequencing data, the genome size of phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 was 44.05, 43.94 kb and XX.XX kb, respectively. The complete genome sequences of the phages were deposited in GenBank under accession number GQ422450, GU595417 and JN225449 respectively. The study of DNA sequence of selected phages showed that UAB_Phi20 has a significantly homology to P22-like phages, UAB_Phi78 was similar to SP6-like phages and UAB_Phi87 has a significant homology to Felix01-like phages. These data are in agreement with those obtained by electron microscopy regarding morphological characterization (Example 5). The analysis of DNA sequences indicated that no virulence genes are encoded in the genomes of the phage selected.
Example 7
In Vitro Killing Assays
S. Typhimurium ATCC14028 and S. Enteritidis LK5 cultures (OD550=0.2) were infected with the bacteriophages selected for the phage cocktail compositions at a multiplicity of infection (relation between the number of infective phages per bacterial viable cell) of 1 pfu/cfu. Cultures were incubated with shaking at 37° C. and both the absorbance of cultures and bacterial enumeration by platting of serial dilutions were monitored.
The viable concentration of S. Typhimurium was reduced 4.4, 3.4, 5.3, 4.3 and 4.4 log10 cfu/ml after infection with each individual phage (UAB_Phi20, UAB_Phi78 and UAB_Phi87), a combination of UAB_Phi20 and UAB_Phi87 (1:1) and with the phage cocktail (UAB_Phi20 and UAB_Phi78 and UAB_Phi87 (1:1:1), respectively (FIG. 4 panel A). In addition, a reduction of 3 log10 cfu/ml was observed after eight hours of treatment with the phage cocktail, which has a proportion 1:1:1 of the three phage selected. A greater effect of phages was seen on S. Enteritidis (FIG. 4 panel B). Thus, the viable concentration of S. Enteritidis suffered a reduction of 5.6, 4, 5.4, 5.9 and 6.5 log10 cfu/ml after treatment with UAB_Phi20, UAB_Phi78, UAB_Phi87, a combination of UAB_Phi20 and UAB_Phi87 (1:1) and the phage cocktail, respectively. In this case, a reduction of 4 log10 cfu/ml was obtained after 8 hours of the infection with the phage cocktail, which has a proportion 1:1:1 of the three phages selected.
The combined infective activity of the three phages into the cocktail achieves a great reduction of Salmonella over the time. This reduction is much better than that achieved by other phage cocktail tested only for 4 hours of phage infection (Toro et al., 2005). The fact that individual phages of the invention were not so efficient to maintain low concentrations of Salmonella over the time justify the use of two or three phages in a bacteriophage cocktail for the purpose of the invention.
Example 8
Experiments in Animal Models
White Leghorn chicken specific pathogen free (SPF) were used to assay the efficacy of the phage cocktail compositions in decreasing the S. Typhimurium ATCC14028 ΩCm concentration in in vivo experiments. In this model, animals are a reservoir of non typhoidal Salmonella. It is expected that animal treatment with phage cocktail diminished the Salmonella concentration in the digestive system of chicks.
White Leghorn chicken specific pathogen free (SPF) fertile eggs from Valo Lohmann Tierzucht (Salamanca, Spain) were used for these studies. Fertile eggs were incubated and hatched in level-3 biocontention facilities for animals (BSL3) of CReSA (Cerdanyola del Valles, Spain). Chicks of all trials were housed in air-filtered isolator cabinets. Feed and water were supplied ad libitum. In all trials, two chicks were euthanized before the experiments to check that they remained free of natural infection of Salmonella. 
Previously to assay the effect of phage cocktail composition on Salmonella in chicks, the residence time of phages in the chick digestive system was determined. To achieve this, chickens were treated with the phage cocktail at a dose of 1010 pfu of each phage/animal. This cocktail was composed by UAB_Phi20, UAB_Phi78 and UAB_Phi87 (1:1:1) at a concentration of 1011 pfu of each phage/ml in MgSO4 10 mM and 0.1 ml was orally administrated. Afterwards, the phages were recovered from cecum by the enrichment procedure (Example 2) till the eight day after phage cocktail administration, but a significant decrease in the phage concentration was detected at the 4 day of phage administration to animals.
In attention to the results above commented, two experiments were addressed to determine the efficacy of the phage cocktail in decreasing the Salmonella concentration in chicks in which the phage cocktail was administrated to animals at a dose of 1010 pfu/animal. This cocktail was composed by UAB_Phi20, UAB_Phi78 and UAB_Phi87 (1:1:1) at a concentration of 1011 pfu of each phage/ml in MgSO4 10 mM and 0.1 ml was orally administrated.
In the first experiment, chicks were colonised with high concentrations of Salmonella (108 cfu/g of cecum contents) and only four doses of the phage cocktail were administrated. To do this, seeder infection of chicks with Salmonella, which simulates natural conditions of infection with this bacterium, was performed by using the methodology described by Fiorentin et al. (2005a). Two groups of thirty one-day chicks each one were housed on separated isolators. Both groups were infected with Salmonella, but only one of them was treated with the bacteriophage cocktail (treatment group). Previously to phage administration, both groups were infected with S. Typhimurium. To achieve this, three animals of each group (control and treatment groups) were orally inoculated with this bacterium at a dose of 108 cfu/animal while the remaining chicks were infected by contact. It has been previously demonstrated that all chicks have been infected with Salmonella by contact after three days of the oral infection of the first three animals. Afterwards, two doses per day of the phage cocktail (1010 pfu/animal) at 4 and 5 days after Salmonella infection were administrated to the animals of the treatment group. Three chicks of each group were euthanized and necropsied at days 0, 1, 2, 8, 15 and 22 post-treatment. Samples of spleen, liver, ileum and cecum were collected. Likewise, cloacal swabs of all remaining birds were also taken at the same days. Samples were subjected to Salmonella detection and phage enumeration. In all cases, samples from tissues were weighed, homogenized and serially diluted in sodium chloride 0.9%. Salmonella enumeration was determined by standard plating onto XLD plates supplemented with chloramphenicol and incubated at 37° C. during 24 h. At the same time, an enrichment procedure was also performed. To do this, tissue homogenates and cloacal swabs were incubated overnight on peptone water at 37° C. during 24 h. Following, 0.2 ml of these cultures were diluted onto 2 ml of Müller-Kauffmann selective broth and incubated at 37° C. for 24 h. Then, 0.1 ml were plated onto XLD conveniently supplemented plates and incubated at 37° C. for 24h for qualitative bacteriology. The administration schedule of the phage cocktail assayed in this trial produced a reduction of llog10 with respect to the control group during the first two days post-treatment (p=0.055) (FIG. 5).
In the second experiment, chicks orally infected with Salmonella one day after to the administration of phage cocktail and nearly simultaneously to treatment with phages. To perform this, three groups of 23 three-day chicks each one were housed on separated isolators and orally infected with Salmonella at a concentration of 105 cfu/animal. One group was the control, the second one was treated with the phage cocktail one day before the Salmonella infection and the third one received the treatment of the phage cocktail simultaneously to bacterial infection. In addition, in the two last groups the phage cocktail was also administered twice a day on days 1, 2 and 3 post-infection and once a day on days 6, 8, 10, 13, 15 and 17 post-infection. Three chicks of each group were euthanized and necropsied on days 1, 2, 6, 8, 10, 13, 15 and 18 after bacterial inoculation. Sampling and culture methods were performed as described before. Similar results were obtained in both cases. Thus, a decrease of approximately 4 log10 was reached till the third day post-treatment and a decrease of 2 log10 was obtained from day 7 (FIG. 6 panel A) or 11 (FIG. 6 panel B) to the end of the experiment (p<0.0001). These Salmonella reduction values over time are better to those reported by other authors (Toro et al., 2005; Atterbury et al., 2007; Bode et al., 2008; Filho et al., 2007).
Results obtained clearly indicated that the oral treatment of animals with phages or phage cocktails was able to reduce significantly the level of Salmonella in the intestinal system of chicks before animals were colonized by this bacterium or when bacterial colonization would be in an initial phase. Furthermore, the administration schedule of phages or phage cocktails with this purpose must be almost daily, because the residence time of phages at the desired concentrations in the intestinal tract of animals is very short.
Example 9
Application of Phage Cocktail to Reduce Salmonella from Animal Skin
In order to simulate a practical way to apply the phage cocktail in animals before slaughter and thus reduce or extinguish the possible contamination on final product, experiments to assess the efficacy of phage cocktail to reduce the concentration of Salmonella in animal skin was designed. The phage cocktail assayed was composed by UAB_Phi20, UAB_Phi78 and UAB_Phi87 (1:1:1). Skin assayed was swine skin pieces (rear flank) purchased from a local butchery.
Skin pieces were experimentally contaminated with S. Typhimurium ATCC14028ΩCm and S. Enteritidis LK5 ΩKm. An inoculum of 106 cfu/ml in 0.9% NaCl was distributed uniformly on each skin piece of approximately 1,500 cm2 (2 μl/cm2). After, skins were dried for 30 min in a laminar flow cabinet. By means of this treatment, we had previously determined that the final concentration of Salmonella was 102 cfu/cm2. After Salmonella contamination, the surface was divided in two equal sections. One of them was treated with phage cocktail and the other was the control. The treatment consisted of spraying the entire surface with a solution of phage cocktail with 1010 pfu of each phage/ml. The control was also sprayed with a solution of MgSO4 10 mM without bacteriophages. Following, pieces were dried during 30 minutes in a laminar flow cabinet. Afterwards, pieces were incubated at 33° C. and samples were taken by swabbing 6 areas of 25 cm2 at different times. The swab (Copan Italia, Brescia) was placed in a tube with 2 ml of buffered peptone water (Merck, Germany). Following, 1 ml was diluted and plated by duplicate on XLD plates, adequately supplemented with chloramphenicol or kanamycin, and incubated at 37° C. for 24 h. At least, a 4 log10 cfu/cm2 and 2.0 log10 cfu/cm2 reduction of S. Typhimurium and S. Enteritidis, respectively, was reached after treatment with the phage cocktail (FIG. 12). In both cases, the reduction of Salmonella colonization was significant (p<0.005).
Results obtained show that the phage cocktail of this invention is able to reduce the concentration of Salmonella in skin surface when they are applied simulating the conditions of pigs in a holding pen in the processing industries.
Example 10
Application of the Phage Cocktail to Food and Food Manufacturing Process to Reduce the Salmonella Populations
Experiments to assess the efficacy of phages and phage cocktails to reduce the concentration of Salmonella in foods and in food manufacturing processes were designed. The phage cocktail assayed was composed by UAB_Phi20, UAB_Phi78 and UAB_Phi87 (1:1:1). Foods assayed were pieces of packed romaine lettuce (Laetuca sativa) (ready to eat) and chicken breasts purchased from a supermarket.
Romaine lettuce samples were experimentally infected with S. Typhimurium ATCC14028ΩCm and S. Enteritidis LK5 ΩKm by submerging the pieces of lettuce in suspensions of those bacteria at a concentration of 107 cfu/ml in 0.9% NaCl on a ratio of 1:10 (w/v) for 5 minutes and after air dried for 15 min in a laminar flow cabinet. By means of this treatment we had previously determined that the final concentration of Salmonella was 104-105 cfu/g of romaine lettuce. After Salmonella contamination, 25 g of lettuce were placed in a Whirl-Pak bag (Nasco, Fort Atkinson, Wis.) containing 100 ml of a phage cocktail (109 pfu of each phage/ml suspension) in sterile distilled water. Treatment of pieces of lettuce with phages was performed at 25° C. during different times and shaking in a vertical shaker at 150 rpm. After phage treatment, for Salmonella enumeration, the suspension was discarded and 100 ml of buffered peptone water (Merck, Germany) was added to each bag and samples stomached at 150 beats for 1 min. Following, 1 ml was serially diluted and plated by duplicate on XLD plates, adequately supplemented with chloramphenicol or kanamycin, and incubated at 37° C. for 24 h. Sterile distilled water instead of phage cocktail was used as negative control in all the assays. In addition, a control without Salmonella contamination and phage treatment was also performed in parallel. A 3.9 log10 cfu/g and 2.2 log10 cfu/g reduction of S. Typhimurium and S. Enteritidis, respectively, was reached after treatment of lettuce pieces with the phage cocktail for 60 min (FIG. 13).
The same methodology was assayed but treating lettuce pieces with the individual phages UAB_Phi20, UAB_Phi78 and UAB_Phi87 at a concentration of 109 pfu/ml.
These experiments showed that UAB_Phi20 was the most effective phage to reduce Salmonella (FIG. 14). Thus, the concentration of Salmonella in lettuce pieces treated with this phage was very close to those of lettuce pieces treated with the bacteriophage cocktail (FIG. 14).
Chicken breasts were cut and experimentally infected with S. Typhimurium and S. Enteritidis by submerging the pieces into suspensions of those bacteria at a concentration of 106 cfu/ml in 0.9% NaCl on a ratio of 1:10 (w/v) for 5 minutes at room temperature and after the suspension was discarded. By means of this treatment we had previously determined that the final concentration of Salmonella was about 104 cfu/g of chicken breast pieces. After Salmonella contamination, 25 g of chicken breast pieces were placed in a Whirl-Pak bag (Nasco, Fort Atkinson, Wis.) containing 100 ml of a phage cocktail (109 pfu of each phage/ml suspension) in saline solution 0.9% on a ratio of 1:10 (w/v) for 5 minutes and after the suspension was discarded. Following the phage treatment, the chicken breast pieces were maintained in the refrigerator at 4° C. during one week. Samples were daily taken and Salmonella concentration was determined as above described. A 3.9 log10 cfu/g and 0.9 log10 cfu/g reduction of S. Typhimurium and S. Enteritidis, respectively, was reached at the 7 day of conservation of the chicken breast pieces at 4° C. after phage cocktail treatment (FIG. 15).
Results obtained show that the phages of this invention and a phage cocktail are able to reduce the concentration of Salmonella in foods when they are applied in a suspension at room temperature during 30-60 min. Phages are also effective in reducing Salmonella in foods conserved at 4° C. during 7 days, after a very short treatment with phages before food refrigeration. In both cases, the reduction of Salmonella colonization was significantly (p<0.0001). These results indicate that phages and phage cocktail can be used for reducing Salmonella in foods and in food manufacturing processes.
Whereas, particular embodiments of the invention have been described herein for purposes of description, it will be appreciated by those skilled in the art that numerous variations of the details may be made without departing from the invention as described in the appended claims.

## Claims

1. A bacteriophage-composition for treating and/or controlling infectious diseases caused by Salmonella comprising at least two of the Salmonella lytic bacteriophages UAB_Phi20, UABPhi78 and UAB_Phi87 and/or parts or products thereof having lytic activity against Salmonella.

2. The bacteriophage-composition according to claim 1, comprising the following three phages: UAB_Phi20, UAB_Phi78 and UAB_Phi87.

3. The composition according to claim 1 which further comprises other Salmonella lytic bacteriophages or parts thereof having lytic activity against Salmonella.

4. The composition according to claim 3 wherein the Salmonella lytic bacteriophages or parts thereof are selected from the Caudovirales order.

5. The composition according to claim 1 wherein when one or more of the bacteriophages and/or parts or products thereof having lytic activity against Salmonella are present, they are in the same proportion.

6. The composition according to claim 1 wherein when one or more of the bacteriophages and/or parts or products thereof having lytic activity against Salmonella are present, they are in a different proportion.

7. The composition according to claim 1 which additionally includes a pharmaceutically acceptable carrier, solvent or pharmaceutically acceptable excipient.

8. The composition according to claim 1 in the form of a suspension, spray, aerosol, injectable and/or orally administrable dosage form.

9. The composition according to claim 1 in the form of an animal feed, drinking water, sanitizer or cleaning solution.

10. A method of controlling Salmonella populations in a solid matrix selected from the group consisting of poultry barns, swine barns, swine pens, poultry pens, crates for animal transportation, slaughterhouses, and production facilities in food processing industries comprising applying a composition comprising at least two of the Salmonella lytic bacteriophages UAB_Phi20, UAB_Phi78 and UAB_Phi87 and/or parts or products thereof having lytic activity against Salmonella to the solid matrix.

11. The method of claim 10 wherein food processing industries are selected from the group consisting of vegetables, meat and eggs industries.

12. A method of treating or controlling Salmonella infection in animals comprising treating with or applying into or onto an animal a bacteriophage- composition according to claim 1.

13. The method of claim 12 wherein the treatment consists of a daily continuous treatment characterized in that the daily dosage regime per animal per day is selected from the range comprising from 109 to 1012 pfu/day per animal.