Bacteriophage with activity against pathogenic strains of Salmonella
Inventors
Dastych, Jarosław • Dziadek, Jarosław • Górecka, Elżbieta • Rumijowska-Galewicz, Anna • Wojtasik, Arkadiusz • Wójcik, Ewelina
Assignees
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Publication Number
US-9476086-B2
Publication Date
2016-10-25
Expiration Date
Abstract
A method for obtaining a strain of bacteriophage specific to a selected strain of bacteria was found as well as bacteriophage strains obtained in this way. Moreover, application of bacteriophages in manufacturing of the preparation for preventing and fighting infections of farm animals, especially poultry, with pathogenic strains of bacteria sensitive to these phages was described.
Core Innovation
The invention relates to a sterile composition comprising one or more strains of bacteriophage selected from PCM F/00069 (strain 8sent1748), PCM F/00070 (strain 8sent65), and PCM F/00071 (strain 3sent1). Each strain is present at a concentration of at least 2.0×10^6 plaque forming units (PFU) per milliliter (ml), the composition does not comprise Salmonella, and the composition is formulated in a microencapsulated dosage form.
The described work addresses the problem of Salmonella infection and the need for a bacteriophage-based formulation suitable for poultry use. The document reports selection and isolation of Salmonella-specific bacteriophages by screening and downstream preparation, including activity screening using resazurin/alamarBlue-based assays. The selected phage strains are deposited and shown to lyse across multiple Salmonella serotypes.
The innovation further focuses on performance and safety characteristics of the sterile, microencapsulated phage composition, including stability in refrigeration for at least the stated storage duration. The document also describes endotoxin (LPS) testing and cytotoxicity evaluation using neutral red uptake in 3T3 cells, and it reports a broiler study indicating reduced Salmonella infection after cessation, together with an observation that the phages do not persist in internal organs.
Claims Coverage
The independent claim covers a sterile, microencapsulated dosage-form bacteriophage composition containing a specific deposited strain set at a defined minimum concentration, while excluding Salmonella. The independent claim is further constrained by dependent claims that add concentration ranges, quantitative stability limits, an endotoxin-free requirement, and selection of microencapsulated solid versus other microencapsulated dosage forms.
Sterile, microencapsulated phage composition without Salmonella
A sterile composition comprising one or more strains of bacteriophage selected from PCM F/00069 (strain 8sent1748), PCM F/00070 (strain 8sent65) and PCM F/00071 (strain 3sent1), formulated in a microencapsulated dosage form, wherein the composition does not comprise Salmonella.
Minimum phage concentration in the composition
Each of the one or more strains of bacteriophage is present in the composition at a concentration of at least 2.0×10^6 plaque forming units (PFU) per milliliter (ml) (PFU/ml).
Defined PFU/ml concentration range
Each of the one or more bacteriophage strains is present at a concentration from 2.0×10^6 PFU/ml to 2.0×10^8 PFU/ml.
Quantified stability under refrigeration storage
The bacteriophage titer decreases by no more than 90% when stored at 4 degrees C for three months.
Endotoxin-free composition
The sterile composition is endotoxin free.
Microencapsulated solid dosage form
The composition is formulated in a microencapsulated solid dosage form.
Overall, the claim set centers on a sterile, microencapsulated dosage-form bacteriophage composition using deposited Salmonella-specific strains (PCM F/00069 8sent1748, PCM F/00070 8sent65, PCM F/00071 3sent1) at at least 2.0×10^6 PFU/ml and optionally within a defined 2.0×10^6 to 2.0×10^8 PFU/ml range. Coverage is further limited by a quantified refrigeration stability requirement, an endotoxin-free constraint, and by specifying a microencapsulated solid dosage form.
Stated Advantages
Reduces Salmonella infection in broiler chickens (reported as about 200-fold reduction one week after cessation).
Does not result in phage persistence in internal organs in the broiler study.
No acute cytotoxicity observed in neutral red uptake assay (3T3 cells) up to the stated tested particle level.
Sterile composition and endotoxin-free composition (as stated by the claims).
Documented Applications
Poultry feed additive use based on selected Salmonella-specific bacteriophages (broiler chickens study reported).
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