ANTIBACTERIAL FORMULATION COMPRISING A MIXTURE OF BACTERIOPHAGES; USE AND METHOD FOR PREVENTING OR TREATING DISEASES CAUSED BY SALMONELLA SPP. IN FARM ANIMALS BY ORAL ADMINISTRATION OF THE FORMULATION

Abstract

An antibacterial formulation having a mixture of bacteriophages with lytic activity against strains of Salmonella spp. and a pharmaceutically and veterinarily acceptable vehicle, pH stabilizer and/or excipients. This formulation is for the prevention and treatment of infectious diseases caused by Salmonella spp. and different serovars; use and method for preventing or treating infectious diseases caused by Salmonella spp. in farm animals by administering the antibacterial formulation to a non-human animal orally.

Claims

1. An antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. comprising: a) an effective amount of specific bacteriophages: Bacteriophage SenM-L8 IDAC deposit 060820-01, Bacteriophage SenM-STM1 IDAC deposit 060820-03, Bacteriophage SenM-STM23 IDAC deposit 060820-04, Bacteriophage SenS-STM47B IDAC deposit 060820-05, Bacteriophage SenM-M7 IDAC deposit 060820-06, and b) an acceptable vehicle, buffer and/or excipients.

2. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein the formulation the bacteriophages are at concentrations of 9×10.sup.6 to 9×10.sup.9 PFU/mL.

3. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein the bacteriophages with respect to the total mass in the formulation are included in the range of 0.2-0.8% w/v.

4. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein in the formulation the acceptable excipients correspond to preservatives of the anionic salts group, the parabens group and the chelators group.

5. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1 wherein in the formulation the vehicle is water.

6. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein the excipients and components with respect to the total mass in the formulation are included according to the following % w/v: TABLE-US-00011 pH stabilizers 0.5-1.5% w/v Preservatives 0.5-1.0% w/v Bacteriophages 0.2-0.8% w/v Water Amount to complete 100% w/v

7. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein it can be formulated as a veterinary formulation.

8. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein it is formulated as a veterinary oral formulation.

9. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein it is formulated as a veterinary formulation for oral administration in liquid or powder form.

10. The antibacterial formulation comprising a mixture of bacteriophages having lytic activity against strains of Salmonella spp. according to claim 1, wherein a it is formulated as an environmental, surface and equipment disinfection liquid formulation.

11. Use of the antibacterial formulation according to claim 1, wherein it is suitable for the prevention and treatment of infectious diseases caused by Salmonella spp.

12. Use of the antibacterial formulation according to claim 1, wherein it serves to prepare a medicament useful for the prevention and treatment of infections produced by Salmonella serovars infantis, typhimurium, Worthington, Mbandaka, Anatum, Livingstone, Manhattan, Bredeney, Agona, I1,4[5]),12:i:-, Sandiego.

13. Use of the antibacterial formulation according to claim 1, wherein to it serves to prepare a medicament useful for the prevention and treatment of infections caused by Salmonella serovars infantis, typhimurium, Worthington, Mbandaka.

14. Use of the antibacterial formulation according to claim 1, wherein to it serves to prepare a medicament useful for the prevention and treatment of infections caused by Salmonella in a non-human or farm animal.

15. Use of the antibacterial formulation according to claim 1, wherein it serves for preparing a medicament useful for the prevention and treatment of Salmonella infections in poultry.

16. Use of the antibacterial formulation according to claim 10, wherein it serves to decrease the bacterial load of Salmonella on surfaces and equipment.

17. Use of the antibacterial formulation according to claim 16, wherein it serves to decrease the bacterial load of Salmonella on poultry breeding, transfer, slaughter and processing surfaces and equipment.

18. (canceled)

19. (canceled)

Description

FIGURES

[0054] FIG. 1: Resistance profile of bacterial strains. a) Acquired resistance of Salmonella enterica strains from the repository (in silico). The resistance classification (Magiorakos et al., 2012) is indicated on the right: (S) Susceptible to all categories, (R) Resistant to between 1 or 2 categories and (MR) Multiresistant, i.e. resistant to 3 or more categories. b) Prevalence of acquired resistance to antibiotic categories of Salmonella enterica isolates. The graph and the first row of the table show the frequency of resistant isolates from an n=119, the following rows indicate the relative frequency of resistance according to serovar.

[0055] FIG. 2: Resistance profile of Salmonella enterica isolates. Describes the resistance profile of Salmonella isolates according to the method of Magiorakos (2011): (S) Susceptible to all categories, (R) Resistant to between 1 or 2 categories and (MR) Multiresistant, i.e. resistant to 3 or more categories. The graph and the first row of the figure indicate the absolute frequency considering the isolates of an n=119 and in the following rows, it is indicated according to the isolate serovar.

[0056] FIG. 3: Comparative analysis of bacteriophage genomes. a) The size, GC % and genomic map of the five bacteriophages are described. The coding regions are represented as bars which were assigned a color according to their biological function and the non-coding regions were assigned only horizontal lines. b) An identity matrix of the five bacteriophages obtained by sequence alignment using Blast is presented. The darker grayscale indicates greater identity in the sequences.

[0057] FIG. 4: Growth inhibition effect of five bacteriophages on repository enteric S. enterica. The graphs depict the curves obtained after measurement of OD600 over the culture time of each bacteriophage in the repository. The black line indicates the control without bacteriophage treatment, the rest of the dotted lines indicate each of the bacterial assays with the different bacteriophages.

[0058] FIG. 5: Bacteriophage activity profile under different acidity conditions. The circles above the white area in each graph represent the mean titer (n=3) and the error bars indicate the standard deviation. The dotted lines indicate the detection limits and the gray area represents the values above and below these limits.

[0059] FIG. 6: Activity profile of bacteriophages at different temperatures. Activity profile of candidate bacteriophages at different temperature conditions. The circles above the white area in each graph represent the average titer (n=3) and the error bars indicate the standard deviation. The dotted lines indicate the detection limits and the gray area represents the values above and below these limits.

[0060] FIG. 7: Host range of the bacteriophage cocktail against the 119 S. enterica isolates. a) Proportion of isolates according to the type of effect: Total Inhibition implies growth inhibition above 85%, Partial Inhibition, between 15% and 85% and Null Effect below 15%. b) Distribution of the type of antimicrobial effect, according to serovar. c) Examples of inhibitory effect on the growth of S. enterica isolates, according to type of effect.

[0061] FIG. 8: Salmonella spp., S. infantis and S. typhimurium load in rectal swabs. The graph presents the average and SEM of the determination of Salmonella load in rectal swab samples, which were analyzed by qPCR on days 28 and 35. a) indicates the load of Salmonella spp. and b) indicates the load of S. infantis. The load is indicated as DNA copies/mL. Gray bars indicate control group samples and black bars indicate treated group samples.

[0062] FIG. 9: Prevalence of Salmonella spp. evaluated by traditional method in samples of shoe covers. The graph shows the prevalence of Salmonella in footwear covers by the traditional method. The number of wards is indicated and the presence of Salmonella is shown in black and the absence of Salmonella in gray.

EXAMPLES

Example 1. Obtaining Salmonella Strains and Preparation of Bacteriophages

1.1 Obtaining Salmonella Strains.

[0063] Salmonella strains were acquired from the repository of the ISP (Instituto de Salud Pública, Chile) and the American Type Culture Collection (ATCC, USA) (Table 1). In addition, strains isolated from footwear covers and cloacal swabs were obtained from different industrial broiler farms in Chile, particularly broiler hatcheries or broiler breeding halls. These samples were analyzed for the presence of Salmonella spp. according to the Screening Method VIDAS® EASY SLM AFNOR BIO 12/16-09/05 or Method for detection of mobile Salmonella spp. in feces and crawling swabs ISO 6579:2002/Amd 1.

TABLE-US-00002 TABLE 1 Salmonella enterica strains acquired from ISP (Chile) and ATCC. Repository Serovar Repository Code Internal ID Typhimurium ISP ISP 3143-11 Typhimurium R_003 Mbandaka ISP ISP 3663-11 Mbandaka R_007 Infantis ATCC ATCC 51741 Infantis R_027

[0064] Of those samples that tested positive for the presence of Salmonella, a saturated culture was prepared and inoculated into a tube containing 5 mL of TSB, 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. In addition, the growth conditions of each of the strains were determined, the linear range and the equation for the quantification of the microbiological titer were identified by measuring OD600. The strain that presented the widest range of quantification is typhimurium R_003, allowing quantification between 2.3×10.sup.7 and 1.1×10.sup.9 CFU/mL, infantis R_027 (between 1.7×10.sup.7 and 5.0×10.sup.8 CFU/mL) and finally, Mbandaka R_007 (between 1.2×10.sup.7 and 4.8×10.sup.8 CFU/mL).

[0065] On the other hand, the results indicate that a total of 119 S. enterica strains were isolated from broiler breeding sectors, most of these isolates (64.7%) were identified as serovar infantis, with typhimurium (19.3%) being the second most prevalent serovar. Worthington, Mbandaka, Anatum and Livingstone serovars were found in a smaller proportion, representing between 1.7% and 4.2% of the total, respectively. The serovars with the lowest frequency (0.9%) detected were Agona, Bredeney, I1,4,[5],12:i:-, Manhattan, Sandiego and Senftenberg, with one isolate of each.

1.2 Preparation of Bacteriophage Suspension.

[0066] Initially, a mixture of bacteriophages was prepared in TSB medium. This mixture was inoculated with a saturated culture of Salmonella spp. strain, which was selected as host. This co-inoculum was incubated for a period of 18 hours at 37° C. with a constant agitation of 200 rpm.

[0067] To purify the bacteriophages from the culture, it was centrifuged at 3200 g for 5 minutes to separate the bacteria and large particles. The supernatant was filtered using a 0.45 μm porosity polyethersulfone (PES) membrane.

[0068] To obtain the viral titer, 15 mL of TSB with agar was placed and incubated at room temperature for a period of 30 minutes. In parallel, 3 mL of TSB with agar was mixed with 1 mL of saturated host bacterial culture and 1 mL of TSB, and poured onto the previously prepared TBS plate.

[0069] Subsequently, serial dilutions of bacteriophage concentrate were prepared in potencies of 10. These dilutions were inoculated onto the TSB plate for 18 hours at 37° C. After the incubation period, the viral microbiological titer was determined by counting lysis plaque forming units (PFU).

Example 2: Determination of Nucleotide Sequences from Bacterial and Viral DNA and their Characterization

2.1. DNA Extraction.

[0070] Bacterial and viral DNA was purified using the Thermo Fisher extraction kit, following the manufacturer's recommendations. DNA quantification was performed by fluorimetry in the Qubit Fluorometer 3.0 kit (Thermo Fisher), with the reagents and parameters recommended by the manufacturer.

2.2. Library Synthesis.

[0071] With the DNA obtained in the previous step, double-stranded DNA libraries were prepared using the Illumina library preparation kit, with paired-end partitions. The size profile of the libraries was verified using the Fragment Analyzer kit (Agilent), according to the manufacturer's instructions. DNA was quantified by fluorimetry using the Qubit Fluorometer 3.0 (Thermo Fisher), according to the manufacturer's instructions.

2.3 Genetic Characterization of Viral DNA and Bacterial DNA.

[0072] A DNA sequence analysis was performed on the samples obtained in point 1.2. For this purpose, bacterial genomic DNA sequencing and analysis of Salmonella isolates was performed.

2.4. Bacterial Genomes Characterization.

[0073] From the bacterial genomes obtained in the previous point, different in silico analyses were performed for typing according to essential gene sequences, identification of virulence factors and resistance genes, and determination of the serovar of the bacteria.

[0074] The analyses allowed the identification of the prevalence of Salmonella enterica serovars in the samples. These results are shown in Table 2.

TABLE-US-00003 TABLE 2 Prevalence of serovars identified in S. enterica strains isolated from broiler houses. No of Isolated from Broiler Serovar chickens Percentage (%) Infantis 77 64,.7 Typhimurium 23 19.3 Worthington 5 4.2 Mbandaka 4 3.4 Anatum 2 1.7 Livingstone 2 1.7 Agona 1 >0.9 Bredeney 1 >0.9 I1, 4, [5], 12: i: - 1 >0.9 Manhattan 1 >0.9 Sandiego 1 >0.9 Senftenberg 1 >0.9

[0075] Genes associated with microbial resistance were identified. The results indicated that the repository strains present genes conferring acquired resistance to aminoglycosides. In addition, Mbandaka R_007 and infantis R_027 exhibited resistance to fluoroquinolones. No predicted genes conferring resistance to the other 13 resistance categories were observed. Because all four strains showed acquired resistance (predicted in silico) to one or two categories, they were classified as resistant bacteria (FIG. 1a). With respect to the isolates, all showed acquired resistance (predicted in silico) to aminoglycosides, of which 82% of the isolates also showed resistance to fluoroquinolones. In addition, the presence of resistance to 9 categories of antibiotics was identified, where most of the isolates had resistance genes to tetracyclines (71%) and phenicols (53%). There was no resistance to first or second generation cephalosporins, carbapenemics, cephamycins or glycylcyclines (FIG. 1b).

[0076] On the other hand, it was possible to observe the presence of differences in the resistance acquired by the different serovars. Resistance to penicillins was observed mainly in serovars infantis and typhimurium (in the latter in a low proportion). Only infantis serovar showed resistance to monobactam and extended-spectrum cephalosporins, with a prevalence of 74%. The only serovars that showed resistance to polymyxins were Senftenberg and Mbandaka (FIG. 1b).

[0077] On the other hand, the isolates showed acquired resistance between 1 and 10 of the 15 antibiotic categories. Of these isolates, 75% were classified (according to the method of Magiorakos et al., 2012) as multidrug resistant (resistance to 3 or more categories), the remaining 25% as resistant (between 1 and 2 categories). None of the isolates showed susceptibility to all categories. In addition, variability was observed in the number of categories between serovars and within the same serovar. In the case of infantis, resistance to between three and ten categories was detected, so that all isolates were classified as multidrug-resistant. In the other serovars, resistance to between one and five categories was identified (FIG. 2).

2.5. Viral Genomes Characterization.

[0078] To characterize the viral genomes, different in silico analyses were performed to describe the type of viral DNA packaging, determine the viral DNA replication cycle, detect the presence of coding sequences for integrases, virulence factors and bacterial resistance genes, and establish the taxonomic identity of the bacteriophages.

[0079] The five bacteriophages obtained correspond to the order Caudovirales, which are characterized by being tailed, over-stranded DNA bacteriophages. Additionally, five bacteriophages (SenM-L8, SenM-M7, SenM-STM1 and SenM-STM23) belong to the Myoviridae family, which are characterized by having long and contractile tails. Finally, the five bacteriophages belong to the subfamily Ounavirinae and the genus Felixonavirus, with the exception of SenS-STM47B, which belongs to the genus Tequintavirus and is characterized by a long, non-contractile tail.

[0080] Bacteriophages SenM-L8, SenM-M7, SenM-STM1 and SenM-STM23 have identical genome size, GC content and genetic structure (FIG. 3a). On the other hand, SenS-STM47B, the only Siphoviridae, has a larger genome size and GC content, and the ordering of its genes is different. The genome map of the bacteriophage genomes shows that there are no integrases or virulence factors, a necessary feature in bacteriophages used in phage therapy. When comparing the genomes of the five bacteriophages, a trend similar to that shown by the genomic map is observed: there is a high identity among the bacteriophages of the Myoviridae family (over 99.991%) and these differ significantly from SenS-STM47B, of the Siphoviridae family (76% identity) (FIG. 3b).

Example 3: In Vitro Lytic Activity on Salmonella Strains and Bacteriophages Characterization

3.1. Evaluation of the Inhibitory Effect on Bacterial Growth.

[0081] A culture of the bacterium of interest was mixed in TSB medium having OD600=0.3, and diluted 1:105 with bacteriophage suspensions to an initial MOI greater than 105 PFU/CFU or by bringing the mixture to a final volume of 200 μL with TSB medium. The mixtures were added to a 96-well plate and incubated for 18 hours at 37° C. using EPOCH 2 spectrophotometry equipment with continuous orbital shaking. The OD600 was recorded every 10 minutes. The antimicrobial effect of the mixture was determined by comparing the ratio of the area under the growth curve in the presence and absence of the bacteriophage. Null effect (N): inhibition up to 15% of growth. Partial inhibition (P): between 15% and 85%. Total inhibition (T): reduction over 85%.

[0082] In this assay, 5 bacteriophages (isolated from wastewater from the city of Santiago, Chile) were identified as having antimicrobial activity against some of the S. enterica repository strains. Bacteriophages present different activity profiles, however, they present a similar range of activity against at least 3 of the 4 bacteria in the repository (Table 3).

TABLE-US-00004 TABLE 3 Host range of 6 bacteriophages isolated from wastewater with lytic activity on Salmonella enterica repository strains. Growth inhibition with respect to bacteria without bacteriophage treatment Typhimurium Mbandaka Infantis Bacteriophage ID (R_003) (R_007) (R_027) Amplitude SenM-L8  36% 100% 12% 4 SenM-M7 100% 100% 93% 3 SenM-STM1 100% 100% 71% 4 SenM-STM23 100% 100% 62% 4 SenS-STM47B 100%  94% 60% 4

[0083] The activity of the repository bacteria is affected by each of these bacteriophages. There is a high percentage inhibition on the growth of Mbandaka R_007 and typhimurium R_003, except in the case of phage SenM-L8 which after approximately 4 hours the growth of typhimurium R_003. The effect on the growth of infantis R027 is varied and in general, the inhibition of Enteritidis R_006 growth is low (up to 39%) (FIG. 4).

[0084] Also, the similarity of the five bacteriophages to others bacteriophages already reported was determined. The bacteriophages of this invention are different from those reported in the state of the art, in the results a maximum similarity of 87.8% was obtained. The bacteriophage that is most similar to SenM-L8, SenM-M7, SenM-STM23 and SenM-STM1 is Salmonella Phage Si3 and the most similar to SenS-STM47B is a bacteriophage from Shigella SS 1 (Table 4).

TABLE-US-00005 TABLE 4 Similarity of five bacteriophages of the invention with respect to previously reported bacteriophages. Closest Bacteriophage bacteriophage Coverage.sup.1 Identity Similarity.sup.2 SenM-L8 Salmonella 92.0% 95.5% 87.8% phage Si3 (NC_041922.1) SenM-M7 Salmonella 92.0% 95.5% 87.8% phage Si3 (NC_041922.1) SenM-STM1 Salmonella 92.0% 95.5% 87.8% phage Si3 (NC_041922.1) SenM-STM23 Salmonella 92.0% 95.5% 87.8% phage Si3 (NC_041922.1) SenS-STM47B Shigella 88.0% 94.4% 83.1% phage SSP1 (NC_047881.1) .sup.1Best alignment result in Blastn's “nr” database, GenBank code in parentheses. .sup.2Similarity defined as the product of the identity calculated on the basis of the coverage of the search sequence.

[0085] Finally, other characteristics of the bacteriophages were predicted and it was observed that they present characteristics that indicate that they are suitable for use in phage therapy, in terms of biological safety. All of them present a lytic replication cycle (essential for phage therapy). No elements related to transduction potential such as integrases, recombination sites, genes of bacterial origin, virulence or resistance genes were found (Table 5)

TABLE-US-00006 TABLE 5 Biosafety of the five bacteriophages. Suitable Replication attL/attR Bacteria Virulence Resistance for phage Bacteriophage cycle Integrase sites gene gene gene therapy SenM-L8 Lytic Absence Absence — — — — SenM-M7 Lytic Absence Absence — — — — SenM-STM1 Lytic Absence Absence — — — — SenM-STM23 Lytic Absence Absence — — — — SenS-STM47B Lytic Absence Absence — — — —

3.2. Evaluation of Bacteriophage Stability Under Acidic Conditions.

[0086] An aliquot of 10 μL of concentrated bacteriophage culture was taken and diluted in 250 μL of acid saline (NaCl 0.9% w/v pH 1, 2, 3, 4, 5, 6 and 7) and incubated for 4 hours at 37° C. After incubation, 750 μL of neutralizing solution (NaHCO.sub.3 0.53% w/v+HCl 12.3 mM) was added. The mixture was then incubated for 20 minutes at 4° C. and the viral titer was determined as described in section 1.2.

[0087] The results of this assay showed that three bacteriophages (SenM-STM1, SenM-M7 and SenM-L8) exhibit stable activity between pH 3 and 7 for a period of 4 hours. In addition, SenM-STM1 showed detectable but reduced antimicrobial activity upon exposure to pH 2 and SenM-STM23 for which reduced activity was detected at pH 1 and 2, being the bacteriophage with the widest range of activity under acidic conditions. On the other hand, SenM-STM47B showed a smaller range of activity, remaining stable between pH 4 and 7. It should be noted that the differences in the level of activity of the bacteriophages are due to the initial concentration of each one in the assay (FIG. 5).

3.3. Evaluation of Bacteriophage Stability at Different Temperatures.

[0088] An aliquot of 10 μL of bacteriophage culture was taken at a concentration of 5×10.sup.7 or higher and diluted in 250 μL of acid saline (NaCl 0.9% w/v pH 5.5). The solution was incubated for a period of 4 hours at −20, 4, 25, 37, 40, 50, 60 and 70° C. After incubation, 750 μL of neutralizing solution (NaHCO.sub.30.53% w/v+HCl 12.3 mM) was added. Subsequently, this solution was incubated at 4° C. for 20 minutes and the viral titer was determined as indicated in point 1.2

[0089] Results indicate that the antimicrobial activity of the bacteriophages remained stable when exposed to temperatures between 5 and 50° C. In addition, SenM-M7 and SenS-STM47B showed reduced activity after exposure to 60° C. Interestingly, SenS-STM47B maintained stable activity at −20° C. It is worth mentioning, that the difference in titer between bacteriophages is due to the fact that the initial concentration of each was different. To detect reduced activity in the SenM-STM1, SenM-STM23 and SenM-L8 bacteriophages, it is necessary to increase the concentration in the assay, approaching the upper limit of detection (FIG. 6).

Example 4: Formulation of Bacteriophages with Lytic Activity on Salmonella enterica Isolates

[0090] The combination of bacteriophages: bacteriophage SenM-L8 (IDAC deposit 060820-01), bacteriophage SenM-STM1 (IDAC deposit 060820-03), bacteriophage SenM-STM23 (IDAC deposit 060820-04), bacteriophage SenS-STM47B (IDAC deposit 060820-05) and bacteriophage SenM-M7 (IDAC deposit 060820-06) are prepared with pharmaceutical and veterinary acceptable excipients, according to the following ratios or concentration ranges (Table 6)

TABLE-US-00007 TABLE 6 Formulation components and proportions. Formulation component Proportion (% w/v) pH stabilizers 0.5-1.5 Preservatives 0.5-1.0 Bacteriophages 0.2-0.8 Water Quantity to complete 100%

Example 5. Evaluation of the Efficacy of the Formulation Comprising the Bacteriophage Mixture on Salmonella enterica Isolates

5.1. Determination of Minimal Inhibitory MOI (MIM).

[0091] The growth inhibitory effect was assessed by mixing bacterial cultures diluted 1:105 with OD.sub.600=0.3 with suspensions of bacteriophages at a MOI.sub.initial of 10.sup.−3, 10.sup.−2, 10.sup.−1, 10.sup.0, 10.sup.1, 10.sup.2, y 10.sup.3 PFU/CFU, or bring the mixture to a final volume of 200 μL with TSB medium. As with the antimicrobial activity assay, the suspensions were transferred to a 96-well plate and incubated for a period of 18 hours at a temperature of 37° C. in EPOCH 2 with continuous orbital shaking, determining OD600 every 10 minutes. The antimicrobial effect of each bacteriophage was determined mathematically by comparing the area under the growth curve in the presence and absence of bacteriophage. The MIM was determined as the minimum MOI evaluated in the assay that presented bacterial growth inhibition over 85% (total inhibition).

[0092] The five bacteriophages were then used in a formulation, considering that each one is unique, suitable for phage therapy and with antimicrobial activity on Salmonella. The results showed that the formulation presents antimicrobial activity on 98.3% of the Salmonella enterica isolates obtained from broiler farms. A total growth inhibition of 60.5% (infantis, typhimurium, Mbandaka, Worthington serovars) and a partial inhibition of 37.8% (Anatum, Livingstone, Manhattan, Bredeney, Agona, I1,4[5]),12:i:-, Sandiego serovars). In only 2 of the 119 isolates (1.7%), one of Senftenberg serovar and one of typhimurium serovar, no effect on bacterial growth was observed (FIGS. 7a and 7b).

[0093] The isolates on which the cocktail had a total growth inhibition effect belong to infantis serovar (75% of these), typhimurium (20%) and all Worthington and Mbandaka isolates. These four serovars are the most prevalent among the strains obtained from broiler houses. For the other serovars, with the exception of Senftenberg, at least partial growth inhibition was observed (FIGS. 7b and 7c).

[0094] In addition, the Minimum Inhibitory MOI (MIM) to achieve total inhibition (above 85%, MIM85) and 50% growth inhibition (MIM50) was determined to be different for each repository strain (Table 7). For infantis R_027 MIM85 occurs at an MOI of 0.1 PFU/CFU, i.e. at a ratio of one bacteriophage per ten bacteria, whereas, to reduce bacterial growth by half, 1,000 times fewer bacteriophages than bacteria were required (MOI 0.001 PFU/CFU). The best performance was achieved against typhimurium R_003 since both MIM values are equivalent and correspond to the lowest concentration tested (MOI=0.001 PFU/CFU). A low MOI of 0.001 PFU/CFU is required to inhibit the growth of Mbandaka R_007 by half, however, a much higher MOI of 1 PFU/CFU is necessary to achieve total inhibition.

[0095] Considering the maximum load of Salmonella in the cecum chicken (9×10.sup.6 copies/cecum), would require, in the worst case scenario (MIM85=1 PFU/CFU) a dose of 9×10.sup.6 PFU of cocktail per chicken to achieve total inhibition of infantis, typhimurium and Mbandaka serovars. This dose is compatible with the concentrations achievable at the bacteriophage production level (over 10.sup.9 PFU/mL per bacteriophage, data not shown).

TABLE-US-00008 TABLE 7 Minimal inhibitory MOI of bacteriophage formulation to inhibit the growth of Salmonella enterica. Repository Strain MOI (PFU/CFU) ID MIM.sub.85 MIM.sub.50 Salmonella enterica sv. 0.1 0.001 Infantis R_027 Salmonella enterica sv. 0.001 0.001 Typhimurium R_003 Salmonella enterica sv. 1 0.001 Mbandaka R_007

Example 6: Evaluation of the Efficacy of Bacteriophages in the Reduction of Salmonella enterica in Broiler Chickens

[0096] This evaluation was carried out on a farm in south-central Chile, consisting of 10 wards, with a history of Salmonella spp. prevalence above 40%.

[0097] The results of these trials are presented below

[0098] Determination of Salmonella Spp. and S. infantis Load in Rectal Swab.

[0099] The bacterial load was determined by qPCR from rectal swabs. The results showed that there are significant differences in the load of Salmonella spp. between the “treated” and “control” conditions, with the load being higher in the control group samples at 28 and 35 breeding (p=0.012431; p=0.032947 respectively) (t-test, alpha=0.05). These results can be seen in Table 8 and FIG. 8a.

TABLE-US-00009 TABLE 8 Salmonella spp. bacterial load from rectal swab samples. Control Treated Average Average Breeding (DNA (DNA day copies/mL) SD n copies/mL) SD n 28 3.75 × 10.sup.6 8.55 × 10.sup.6 53 1.28 × 10.sup.6 3.34 × 10.sup.6 98 35 3.76 × 10.sup.6 5.05 × 10.sup.6 48 1.84 × 10.sup.6 4.83 × 10.sup.6 83

[0100] The load of Salmonella enterica serovar infantis showed significant differences between the “control” and “treated” groups, with the “control” group showing a higher load on day 28 (p=0.024505) than the “treated” group (t-Test, alpha=0.05). The samples on day 35 of breeding showed no differences between the study groups (Table 9; FIG. 8b).

TABLE-US-00010 TABLE 9 Bacterial load of Salmonella enterica serovar Infantis from rectal swab samples. Control Treated Average Average Breeding (DNA (DNA day copies/mL) SD n copies/mL) SD n 28 7.82 × 10.sup.6 1.25 × 10.sup.7 40 3.47 × 10.sup.6 8.31 × 10.sup.6 81 35 4.54 × 10.sup.6 5.36 × 10.sup.6 42 2.82 × 10.sup.6 6.04 × 10.sup.6 55

[0101] Determination of Prevalence of Salmonella Spp. by the Traditional Method of Shoe Covers.

[0102] On day 27-29 of breeding, footwear covers were sampled and analyzed by the ISO 6570:2002/Amd1:2007 method. The results indicate that the prevalence of Salmonella spp. is lower in the “treated” group when compared to the “control” group (FIG. 9). This means that the treatment decreased the prevalence of Salmonella spp. when detected by the Traditional Method.

REFERENCES

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