RU2603730C1 - Method of producing bacteriophage - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to production of a bacteriophage. Disclosed method involves inoculation of bacterial cell suspension in the titre of 10⁸-10¹² CFU/ml on a dense nutrient medium with the layer thickness from 3 to 25 mm and cultivation with no foreign microflora, when the air layer thickness over the surface of the dense nutrient medium is from 5 to 50 mm and at optimum temperature for a host strain culture growth. In 30-120 minutes after the beginning of culturing every 30-60 minutes smears are taken from the surface of the dense nutrient medium, coloured with an intercalating dye and microscoped. Cultivation is stopped when reaching at least 10 % in relation to the total amount of cells of the host cell fraction strain with heterogeneous fluorescent cytoplasm staining. Then the obtained lawn of the host strain culture is inoculated with a mother bacteriophage in the titre of 10⁷-10¹⁰ BFU/ml. Cultivated for 13-15 hours with no foreign microflora, at the air layer thickness over the surface of the dense nutrient medium from 5 to 50 mm and at optimum temperature for the bacteriophage strain culture growth. Obtained a phage lysate when suspending, sucked off the phage lysate into a sterile container and cleaned. Invention provides reaching a stable high titre of bacteriophage (10¹²-10¹⁴ BFU/ml) when producing phage lysates both at varying formulations of nutrient media and increasing the span variation of values of culturing the host strain and the bacteriophage.

EFFECT: invention can be used in biotechnology for obtaining products containing bacteriophages.

9 cl

Description

The invention relates to microbiology and can be used in biotechnology to obtain products containing bacteriophages.

Bacteriophages are viruses characterized by a specific ability to selectively infect bacterial cells belonging to the same strain or antigenically homologous strains of the same species or genus, followed by lysis (after intracellular replication) of the host cell — virulent phages, or by integration into the bacterial genome with the formation of lysogens - moderate phages (Adams N.M. Bacteriophages. - New York: Interscience Publishers, Inc., London: Interscience Publishers ltd., 1959. - 592 p.). According to numerous literature data, phages can be used as natural antimicrobial agents to combat bacterial infections in humans, animals, and crops (Alisky J., Iczkowski K., Rapoport, A., Troitsky N. Bacteriophages show promise as antimicrobial agents / / The Journal of infection. - 1998. - Vol. 36, No. 1. P. 5-15; Brussow H. Phage therapy: the Escherichia coli experience // Microbiology. - 2005. - Vol. 151, pt. 7. - P. 2133-2140; Bondarenko BM The clinical effect and the rational use of therapeutic bacteriophages in medical practice // Appendix to the Journal of Infectology. - 2011. - T. 3, No. 3. - P. 15-19). A number of authors admit the possibility of the practical use of bacteriophages as a means of decontamination in the food industry as a safe and environmentally friendly alternative to chemical and physical methods for eliminating bacteria (Greer GG Bacteriophage control of foodborne bacteria // Journal of food protection. - 2005. - Vol. 68, No. 5. - P. 1102-1111; Lang LH FDA approves use of bacteriophages to be added to meat and poultry products // Gastroenterology. - 2006. - Vol. 131, No. 5. - P. 1370-1372; Aleshkin AB, Zeigarnik MB Possibilities of using bacteriophages as probiotic decontamination agents in nutrition // B Requests Diet -. 2012. - V. 2, №4 -. pp 24-34).

One of the priority indicators for the production of phage-containing products is the production of phagolysates with an initially high titer of bacteriophages (Goldfarb D.M. Bacteriophagy. - M.: Medgiz, 1961. - P. 17-19).

A well-known method proposed for various bacteriophages for producing phagolysates with a high titer of bacteriophages is the cultivation of bacteria of the host strain with a bacteriophage in a dense nutrient medium, followed by the preparation of a suspended phagolysate from the surface of a dense nutrient medium (SU 64612, 04/30/1945; Kuzmin N.A., Komarov B.A. A method for the rapid production of anthrax phages in high concentrations // Laboratory. - 1967. - No. 12. - S. 741-743).

The main disadvantages of these analogues of the claimed invention are the lack of versatility of the method for producing products using various bacteriophages, the insufficient stability of the titer of bacteriophages in the phagolysate, and the absence or inadequacy of purification of the phagolysate for the use of phage-containing products in the food industry upon receipt of the phagolysate with a titer of bacteriophage 10 ¹² -10 ¹⁴ PFU / ml

Also known analogue is a method for producing concentrated phage preparations, which consists in seeding on a plate with agar a mixture of a concentrated culture of bacteria and phage in an amount sufficient to obtain a continuous lysis. After incubation at 37 ° C for 12-18 hours, 3-5 ml of broth is poured into cups and left for 20 minutes. Then the broth is drained and centrifuged. The supernatant is aspirated. According to the prototype, a phage prepared in this way may contain 10 ¹¹ -10 ¹² particles per 1 ml (Goldfarb D.M. Bacteriophagy. - M.: Medgiz, 1961. - P. 18).

The main disadvantages of this analogue are the lack of versatility of the method for producing products using various bacteriophages, the insufficient stability of the titer of bacteriophages in the phagolysate and the inadequate purification of the phagolysate for the use of phage-containing products in the food industry when obtaining purified phagolysate with a bacteriophage titer of 10 ¹² -10 ¹⁴ PFU / ml.

The closest analogue prototype is a method for producing a bacteriophage, according to which the bacterial culture of the host strain in a titer of 10 ⁸ -10 ⁹ CFU / ml is seeded in a culture vessel on a beveled dense nutrient medium with a layer thickness of 10 mm to 25 mm, cultured for 3-3.5 hours at the optimum temperature for the growth of the culture of the host strain, then the uterine bacteriophage in the titer of 10 ⁵ -10 ⁶ PFU / ml is sown on the obtained culture law of the host strain, the culture vessel is sealed, cultured for 13-15 hours at the optimum temperature for the growth of the culture of the bacteriophage strain and the thickness of the air layer above the surface of the dense nutrient medium from 25 mm to 40 mm, a phagolysate is obtained by suspending the bacteriophage from the surface of the dense nutrient medium with physiological saline or buffer solution with a pH of 7.0-7.2 in an amount 0.04-0.045 ml per 1 cm ^{2 of the} surface of a dense nutrient medium, the phagolysate is sucked off in a sterile container, chloroform is added, it is kept for 30-45 minutes with continuous shuttling, centrifuged for 30-45 minutes at 5000- 6000 rpm, the supernatant is sterilized by filtration through a filter with a pore diameter of 0.2-0.22 μm and the filtrate obtained is passed through a column containing an agent affinity for endotoxin (RU 2525141, 08/10/2014). After centrifugation, you can mix the supernatant containing bacteriophages, then sterilize the mixture of supernatant by filtration through a filter with a pore diameter of 0.2-0.22 microns and pass the obtained filtrate through a column containing an agent affinity for endotoxin. As a vessel for cultivation, you can use a glass microbiological mattress.

The main disadvantage of the prototype is the dependence of the stability of achieving a high titer of the bacteriophage upon receipt of the phagolysate on compliance with both the constancy of the formulation of the nutrient medium and the small range of variation in the values of cultivation of the host strain and the bacteriophage, which complicates the industrial application of the known method for producing a bacteriophage using a dense nutrient medium.

The main objective of the claimed invention is to ensure the stability of achieving a high titer of the bacteriophage (10 ¹² -10 ¹⁴ PFU / ml) upon receipt of phagolysates under conditions of changing both the formulations of the nutrient media and increasing the magnitude of the variation in the values of cultivation of the host strain and bacteriophage.

The problem is solved in that a suspension of bacterial cells of a rapidly growing host strain in a titer of 10 ⁸ -10 ¹² CFU / ml is seeded in a culture vessel on a solid nutrient medium with a layer thickness of 3 mm to 25 mm, cultivated in the absence of extraneous microflora, with a thickness a layer of air above the surface of a dense nutrient medium from 5 mm to 50 mm and at the optimum temperature for the growth of the culture of the host strain, 30-120 minutes after the start of cultivation every 30-60 minutes from the surface of a dense nutrient medium make smears that stain the inter with an aliasing dye and microscopy under a fluorescence microscope, the cultivation of the host strain is stopped when at least 10% of the total number of cells of the cell fraction with heterogeneous staining of the fluorescent cytoplasm is reached, namely, paired, pole-shaped cells in the form of rods with predominant cytoplasmic fluorescence contiguous poles or cells of spherical and ellipsoidal shape with a weakly fluorescent central section, then on the obtained lawn of the culture of the host strain, sowing a uterine bacteriophage in a titer of 10 ⁷ -10 ¹⁰ PFU / ml is cultured, cultured for 13-15 hours in the absence of extraneous microflora, with a thickness of the air layer above the surface of a dense nutrient medium from 5 mm to 50 mm and at an optimal temperature for the growth of a culture of a bacteriophage strain prepared by slurrying fagolizat bacteriophage surface solid nutrient medium or saline buffer solution with a pH of 7.0-7.4 in an amount of 0.04-0.05 ml per 1 cm ² surface of a dense nutrient medium obtained fagolizat aspirated into a sterile cm spine and purified. After the phagolysate is aspirated into a sterile container, it can be cleaned by sterilizing filtration through a filter with a pore diameter of 0.2-0.45 microns. After the phagolysate is aspirated into a sterile container, it can be cleaned by centrifugation for 30-60 minutes at 3000-6000 rpm and sterilizing filtration of the supernatant through a filter with a pore diameter of 0.2-0.45 microns. After the phagolysate is aspirated into a sterile container, it can be cleaned by adding chloroform, keeping it for 15-45 minutes with continuous shuttling, centrifuging for 30-60 minutes at 3000-6000 rpm and sterilizing filtration of the supernatant through a filter with a pore diameter of 0.2 -0.45 microns. After the phagolysate is aspirated into a sterile container, it can be cleaned by filtration through a filter with a pore diameter of 0.45-1.0 μm and sterilizing filtration of the obtained filtrate through a filter with a pore diameter of 0.2-0.45 μm. After sterilizing filtration, the filtrate obtained can be passed through a column containing an endotoxin affinity agent. Before sterilizing filtration, you can mix liquids containing bacteriophages, and then sterilize the mixture of liquids by filtration through a filter with a pore diameter of 0.2-0.45 microns. After sterilizing filtration, the filtrate obtained can be passed through a column containing an endotoxin affinity agent. As a vessel for cultivation, you can use a glass microbiological mattress.

Purification of the phagolysate is intended primarily to remove living non-lysed cells of the host strain. In addition, purification of the phagolysate allows you to get rid of fragments of lysed cells of the host strain, exopolysaccharides, toxins and other derivatives and metabolites of the host strain. The purification of the phagolysate can be carried out both in one step and in several steps.

The term "rapidly growing host strain" refers to bacterial strains whose colonies or continuous growth can be detected visually after 6-48 hours of cultivation in a solid nutrient medium used in the cultivation of bacteriophages.

For staining smears can be used any DNA staining intercalating dyes, the range of wavelengths of excitation in which is suitable for fluorescence microscopy. As such intercalating dyes, for example, ethidium bromide (Mansour JD, Schram JL, Schulte, T.N. Fluorescent staining of intracellular and extracellular bacteria in blood // Journal of Clinical Microbiology. - 1984. - Vol. 19, No. 4. - P. 453-456), SYBR Green I (Noble RT, Fuhrman JA Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria // Aquatic Microbial Ecology. - 1998. - Vol. 14. - P. 113-118; Patel A., Noble RT, Steele JA, Schwalbach MS, Hewson I., Fuhrman JA Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I // Nature Protocols. - 2007. - Vol. 2 , No. 2. - P. 269-276), SYBR Gold (Shibata A., Goto Y., Saito H., Kikuchi T., Toda T., Taguchi S. Comparison of SYBR Green I and SYBR Gold stains for enumerating bacteria and viruses by epifluorescence microscope opy // Aquatic Microbial Ecology. - 2006. - Vol. 43. - P. 223-231), SYBR Safe (Clauβ M., Springorum AC, Hartung J. Comparison of different fluorescence and non-fluorescence staining techniques for rapid detection of airborne micro-organisms collected on room temperature vulcanizing (RTV) silicones from generated aerosols and from ambient air // Aerosol Science and Technology. - 2012. - Vol. 46, No. 7. - P. 818-827) and others.

As a method for staining a smear with an intercalating dye, any of the options described in the literature (including in the above literature) that provide sufficient fluorescence of bacterial cell cytoplasm for the implementation of the claimed method can be used.

The authors of the claimed method suggest preparing, staining smears and microscopy as follows: a drop of 5-20 μl of an aqueous solution of intercalating dye is applied to the surface of a fat-free glass slide at a concentration exceeding 50-500 times the concentration of the same intercalating dye used for DNA staining with horizontal agarose gel electrophoresis, then a sterile bacteriological loop takes material from the surface of a dense nutrient medium and grind dissolved in a drop of an intercalating dye, allow to dry smear, a smear on the dried surface is applied a drop of non-fluorescent immersion oil and fluorescent microscopy performed with increasing least × 1000 using excitation filter that transmits light in wavelength ranges that are suited to the particular used intercalating dye.

As a result of our first research, we determined a new original distinguishing feature that provides a solution to the problem (stability of achieving a high titer of the bacteriophage (10 ¹² -10 ¹⁴ PFU / ml) when receiving phagolysates under conditions of changing both the composition and quality of the nutrient medium ingredients and increasing the magnitude of the variation in the values of the cultivation indicators of the host strain and bacteriophage) - the criterion for the termination of cultivation of a rapidly growing host strain based on a fluorescence microscope and smears stained with intercalating dye from the surface of a dense nutrient medium — achieving at least 10%, relative to the total number of cells, the proportion of cells with heterogeneous staining of the fluorescent cytoplasm, namely paired, contacting pole-shaped cells in the form of rods with predominant fluorescence of the cytoplasm from the side of the contacting poles or cells spherical and ellipsoidal in shape with weakly fluorescent central section. Based on the use of this new distinctive feature, an increased range of variation in the values of cultivation of the host strain and the uterine bacteriophage (including the titers of the seeded suspension of bacterial cells of the host strain and the seeded uterine bacteriophage, the thickness of the dense nutrient medium layer, the thickness of the air layer above the dense nutrient surface was established medium) with the expansion of the list of formulations of nutrient media, including ingredients from various manufacturers.

From the patent technical literature and the practice of manufacturing phage-containing products, it is not known about the method for producing a bacteriophage, including culturing the bacterial cells of the host strain and the bacteriophage in a dense nutrient medium in the absence of extraneous microflora, obtaining a phagolysate by suspending the bacteriophage from the surface of a dense nutrient medium, and also purifying the phagolysate by sedimentation and / or filtration, which would be identical to the claimed.

Hence, the conclusion about the conformity of the proposed solution to the criterion of "novelty" is legitimate.

The above set of essential features is necessary and sufficient to obtain a technical result — to ensure the stability of achieving a high titer of the bacteriophage (10 ¹² -10 ¹⁴ PFU / ml) when receiving phagolysates under conditions of changing both the formulations of the nutrient media and the increase in the range of variation of the values of strain cultivation host and bacteriophage.

There is a causal relationship between the existing features and the problem to be solved, where each feature is necessary and affects the receipt of a technical result, and together the features taken are sufficient to obtain it. The conclusion that the claimed technical solution meets the criterion of "inventive step" is legitimate.

The proposed method can be implemented repeatedly using its essential features, which means that the claimed technical solution meets the criterion of "industrial applicability".

The invention is tested in obtaining variants of purified phagolysate containing various bacteriophages. The results of this testing are given below. Moreover, the examples of bacteriophage production show a specific implementation of the claimed invention, but do not limit the scope of claims of the claims of the claimed invention.

Example 1. To obtain a bacteriophage active against Salmonella enterica serovar Enteritidis, the bacterial culture of the host strain Salmonella enterica serovar Enteritidis was seeded in a titer of 10 ⁸ CFU / ml in a culture vessel on a solid nutrient medium (GMF agar) with a layer thickness 3 mm.

The host strain was cultured in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium 5 mm thick and at an optimum temperature for its growth (+ 37 ° C). 30 minutes after the start of cultivation, every 30 minutes smears were made from the surface of the solid nutrient medium, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when reaching 10% with respect to the total number of cells, the fraction of paired, contacting poles of stick-shaped cells with predominant fluorescence of the cytoplasm from the side of the contacting poles. Then, the uterine bacteriophage in the titer of 10 ⁷ PFU / ml was sown on the obtained lawn of the Salmonella enterica culture serovar Enteritidis and cultivated for 13 hours in the absence of extraneous microflora, with a thickness of the air layer above the surface of a dense nutrient medium of 5 mm and at the optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with physiological saline in an amount of 0.04 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by sterilizing filtration through a filter with a pore diameter of 0.2 μm.

The obtained purified phagolysate in the form of a filtrate contained bacteriophages in a titer of 10 ¹² PFU / ml in the absence of bacteria and ingredients of the used nutrient medium.

Example 2. To obtain a bacteriophage active against Escherichia coli O104: H4, a bacterial culture of a non-pathogenic host strain - Escherichia coli K12 C600 - in a titer of 10 ¹⁰ CFU / ml was seeded in a culture vessel on a solid nutrient medium (meat-peptone agar) with a layer thickness of 10 mm.

The host strain was cultured in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium 15 mm thick and at an optimum temperature for its growth (+ 37 ° C). 60 minutes after the start of cultivation, smears were made from the surface of a solid nutrient medium every 45 minutes, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when reaching 16% with respect to the total number of cells, the fraction of paired, contacting poles of stick-shaped cells with predominant fluorescence of the cytoplasm from the side of the contacting poles. Then, the uterine bacteriophage in the titer of 10 ⁹ PFU / ml was sown on the obtained lawn of the culture of Escherichia coli K12 C600 and cultivated for 14 hours in the absence of extraneous microflora, with a thickness of the air layer above the surface of a dense nutrient medium of 15 mm and at the optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with a buffer solution with a pH of 7.0 in the amount of 0.045 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by centrifugation for 30 min at 6000 rpm and sterilizing filtration through a filter with a pore diameter of 0.22 μm.

The obtained purified phagolysate in the form of a filtrate contained bacteriophages in a titer of 10 ¹³ PFU / ml in the absence of bacteria and ingredients of the used nutrient medium.

Example 3. To obtain a bacteriophage active against Staphylococcus aureus, the bacterial culture of the host strain, Staphylococcus aureus, in a titer of 10 ¹² CFU / ml was seeded in a glass microbiological mattress on a solid nutrient medium (LB agar) with a layer thickness of 25 mm.

The host strain was cultured in the absence of extraneous microflora, with an air layer thickness above the surface of a dense nutrient medium of 25 mm and at an optimum temperature for its growth (+ 37 ° C). 120 minutes after the start of cultivation, smears were made from the surface of a solid nutrient medium every 60 minutes, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when 55% of the total number of cells was reached, the proportion of spherical and ellipsoidal cells with a weakly fluorescent central section. Then, the uterine bacteriophage in the titer of 10 ¹⁰ PFU / ml was sown on the obtained lawn of the culture of Staphylococcus aureus and cultivated for 15 hours in the absence of extraneous microflora, with a thickness of the air layer above the surface of a dense nutrient medium of 25 mm and at the optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with a buffer solution with a pH of 7.2 in an amount of 0.05 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by adding chloroform to it, keeping it for 15 minutes with continuous shuttling, centrifuging for 60 minutes at 3000 rpm and sterilizing filtration through a filter with a pore diameter of 0.22 μm.

The obtained purified phagolysate in the form of a filtrate contained bacteriophages in a titer of 10 ¹⁴ PFU / ml in the absence of bacteria and ingredients of the used nutrient medium.

Example 4. To obtain a bacteriophage active against Salmonella enterica Typhimurium serovar, a bacterial culture of the host strain Salmonella enterica Typhimurium serovar in a titer of 10 ⁹ CFU / ml was seeded in a culture vessel on a solid nutrient medium (LB agar) with a layer thickness of 10 mm

The host strain was cultured in the absence of extraneous microflora, with a layer of air above the surface of a dense nutrient medium of 40 mm and at an optimum temperature for its growth (+ 37 ° C). 30 minutes after the start of cultivation, every 45 minutes smears were made from the surface of the solid nutrient medium, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when reaching 25% with respect to the total number of cells, the fraction of paired, contacting poles of stick-shaped cells with predominant fluorescence of the cytoplasm from the side of the contacting poles. Then, the uterine bacteriophage in the titer of 10 ⁸ PFU / ml was seeded on the obtained lawn of the Salmonella enterica culture serovar Typhimurium and cultivated for 14 hours in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium of 40 mm and at an optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with a buffer solution with a pH of 7.4 in an amount of 0.045 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by filtration through a filter with a pore diameter of 0.8 μm, sterilizing filtration through a filter with a pore diameter of 0.22 μm and passing the obtained filtrate through a column containing an endotoxin affinity agent.

The resulting phagolysate in the form of an eluate contained bacteriophages in a titer of 10 ¹² PFU / ml in the absence of bacteria, ingredients of the nutrient medium used and at an endotoxin concentration of less than 50 U / ml.

Example 5. To obtain a bacteriophage active against Pseudomonas aeruginosa, a bacterial culture of the host strain, Pseudomonas aeruginosa, in a titer of 10 ¹⁰ CFU / ml was seeded in a culture vessel on a solid nutrient medium (GMF agar) with a layer thickness of 10 mm.

The host strain was cultured in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium 50 mm thick and at an optimum temperature for its growth (+ 37 ° C). 30 minutes after the start of cultivation, every 30 minutes smears were made from the surface of the solid nutrient medium, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when reaching 14% with respect to the total number of cells, the fraction of paired, contacting poles of stick-shaped cells with predominant fluorescence of the cytoplasm from the side of the contacting poles. Then, uterine bacteriophage in the titer of 10 ⁹ PFU / ml was sown on the obtained grass lawn of Pseudomonas aeruginosa culture and cultured for 15 hours in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium of 50 mm and at an optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with a buffer solution with a pH of 7.3 in an amount of 0.05 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by adding chloroform to it, keeping it for 45 minutes with continuous shuttling, centrifuging for 45 minutes at 6000 rpm, sterilizing by filtration through a filter with a pore diameter of 0.45 μm and passing the filtrate through a column containing an endotoxin affinity agent.

The resulting phagolysate in the form of an eluate contained bacteriophages in a titer of 10 ¹² PFU / ml in the absence of bacteria, ingredients of the nutrient medium used and at an endotoxin concentration of less than 50 U / ml.

Example 6. To obtain a bacteriophage active against Staphylococcus aureus, a bacterial culture of the host strain, Staphylococcus aureus, in a titer of 10 ¹² CFU / ml was seeded in a culture vessel on a solid nutrient medium (GRM agar) with a layer thickness of 15 mm.

The host strain was cultured in the absence of extraneous microflora, with a layer of air above the surface of a dense nutrient medium of 35 mm and at an optimum temperature for its growth (+ 37 ° C). 60 minutes after the start of cultivation, smears were made from the surface of a solid nutrient medium every 60 minutes, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when 55% of the total number of cells was reached, the proportion of spherical and ellipsoidal cells with a weakly fluorescent central section. Then, the uterine bacteriophage in the titer of 10 ⁹ PFU / ml was sown on the obtained lawn of the culture of Staphylococcus aureus and cultivated for 15 hours in the absence of extraneous microflora, with a thickness of the air layer above the surface of a dense nutrient medium of 35 mm and at the optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with physiological saline in an amount of 0.045 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by centrifugation for 40 min at 5000 rpm.

To obtain a bacteriophage active against Listeria monocytogenes, a bacterial culture of a non-pathogenic host strain, Listeria innocua, in a titer of 10 ¹¹ CFU / ml was seeded into a culture vessel on a beveled solid nutrient medium (meat-peptone agar with glucose added) with a layer thickness of 17 mm

The host strain was cultured in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium 30 mm thick and at an optimum temperature for its growth (+ 37 ° C). 60 minutes after the start of cultivation, smears were made from the surface of a solid nutrient medium every 30 minutes, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when reaching 12% with respect to the total number of cells, the fraction of paired, contacting poles of stick-shaped cells with predominant fluorescence of the cytoplasm from the side of the contacting poles. Then, the uterine bacteriophage in the titer of 10 ¹⁰ PFU / ml was sown on the obtained lawn of the Listeria innocua culture and cultivated for 14 hours in the absence of extraneous microflora, with an air layer thickness above the surface of a dense nutrient medium of 30 mm and at the optimum temperature for the growth of the culture of the bacteriophage strain 24 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with a buffer solution with a pH of 7.1 in the amount of 0.045 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by adding chloroform to it, keeping it for 30 minutes with continuous shuttling, centrifuging for 45 minutes at 4500 rpm.

The supernatants of the two phagolysates obtained were mixed.

The supernatant mixture was purified by sterilizing filtration through a filter with a pore diameter of 0.22 μm.

The obtained purified mixture (cocktail) of phagolysates in the form of a filtrate contained a staphylococcal bacteriophage in a titer of 10 ¹³ PFU / ml and a listeriose bacteriophage in a titer of 10 ¹² PFU / ml in the absence of bacteria and ingredients of the used nutrient medium.

Example 7. To obtain a bacteriophage active against Salmonella enterica serovar Infantis, a bacterial culture of the host strain Salmonella enterica serovar Infantis was seeded in a titer of 10 ⁹ CFU / ml in a culture vessel on a solid nutrient medium (meat-peptone agar) with a thickness layer of 10 mm.

The host strain was cultured in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium 15 mm thick and at an optimum temperature for its growth (+ 37 ° C). 30 minutes after the start of cultivation, every 30 minutes smears were made from the surface of the solid nutrient medium, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when reaching 17% with respect to the total number of cells, the fraction of paired, contacting poles of stick-shaped cells with predominant fluorescence of the cytoplasm from the side of the contacting poles. Then, the uterine bacteriophage in the titer of 10 ⁸ PFU / ml was sown on the obtained lawn of the Salmonella enterica culture serovar Infantis and cultivated for 13 hours in the absence of extraneous microflora, with an air layer thickness above the surface of a dense nutrient medium of 15 mm and at the optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with physiological saline in an amount of 0.04 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by filtration through a filter with a pore diameter of 1.0 μm.

To obtain a bacteriophage active against Escherichia coli O157: H7, the bacterial culture of the host strain - Escherichia coli K12 C600 - in a titer of 10 ¹⁰ CFU / ml was seeded in a culture vessel on a solid nutrient medium (LB agar) with a layer thickness of 20 mm.

The host strain was cultured in the absence of extraneous microflora, with an air layer above the surface of a dense nutrient medium 30 mm thick and at an optimum temperature for its growth (+ 37 ° C). 60 minutes after the start of cultivation, smears were made from the surface of a solid nutrient medium every 30 minutes, which were stained with an intercalating dye and microscopied under a fluorescence microscope. The cultivation was stopped when reaching 25% with respect to the total number of cells, the fraction of paired, contacting poles of stick-shaped cells with predominant fluorescence of the cytoplasm from the side of the contacting poles. Then, the uterine bacteriophage in the titer of 10 ⁸ PFU / ml was sown on the obtained lawn of the culture of Escherichia coli K12 C600 and cultivated for 13 hours in the absence of extraneous microflora, with a layer of air above the surface of a dense nutrient medium of 30 mm and at the optimum temperature for the growth of the culture of the bacteriophage strain (+ 37 ° C).

The phagolysate was obtained by suspending the bacteriophage from the surface of a dense nutrient medium with physiological saline in an amount of 0.045 ml per 1 cm ^{2 of the} surface of a dense nutrient medium.

Then the phagolysate was aspirated into a sterile container and it was purified by filtration through a filter with a pore diameter of 0.45 μm.

The filtrates of the two phagolysates obtained were mixed.

The mixture of filtrates was purified by sterilizing filtration through a filter with a pore diameter of 0.22 μm and passing the obtained filtrate through a column containing an endotoxin affinity agent.

The obtained purified mixture (cocktail) of phagolysates in the form of an eluate contained a salmonella bacteriophage in a titer of 10 ¹³ PFU / ml and a coliphage in a titer of 10 ¹² PFU / ml in the absence of bacteria, ingredients of the nutrient medium used and at an endotoxin concentration of less than 50 EU / ml.

Claims (9)

1. A method of producing a bacteriophage, including culturing the bacterial cells of the host strain and the bacteriophage in a dense nutrient medium in the absence of extraneous microflora, obtaining a phagolysate by suspending the bacteriophage from the surface of a dense nutrient medium, as well as purifying the phagolysate by sedimentation and / or filtration, characterized in that the suspension bacterial cells of a rapidly growing host strain in a titer of 10 ⁸ -10 ¹² KOE / ml are seeded in a culture vessel on a solid nutrient medium with a layer thickness of 3 mm to 25 mm, cult they are tivited in the absence of extraneous microflora, with a thickness of the air layer above the surface of the dense nutrient medium from 5 mm to 50 mm and at the optimum temperature for the growth of the culture of the host strain, 30-120 min after the start of cultivation every 30-60 min from the surface of the dense nutrient medium smears are made, which are stained with an intercalating dye and microscopied under a fluorescence microscope; the cultivation of the host strain is stopped when at least 10% of the total number of cells in the proportion of cells with a heterogeneous environment is reached ashivaniem fluorescent cytoplasm, namely pair, contacting the cell poles in the form of rods with the preferred fluorescence of the cytoplasm from the adjoining poles or spherical and ellipsoidal cells with weakly fluorescent central section, then the host strain obtained culture lawn seeded uterine bacteriophage at a titer of ¹⁰ July - 10 ¹⁰ PFU / ml, cultivated for 13-15 hours in the absence of extraneous microflora, with a thickness of the air layer above the surface of a dense nutrient medium from 5 mm to 50 mm and with op at the optimum temperature for the growth of the culture of the bacteriophage strain, a phagolysate is obtained by suspending the bacteriophage from the surface of a dense nutrient medium with physiological solution or buffer solution with a pH of 7.0-7.4 in an amount of 0.04-0.05 ml per 1 cm ^{2 of the} surface of a dense nutrient medium , the resulting phagolysate is aspirated into a sterile container and cleaned. 2. The method according to p. 1, characterized in that after the suction of the phagolysate in a sterile container, it is cleaned by sterilizing filtration through a filter with a pore diameter of 0.2-0.45 microns. 3. The method according to p. 1, characterized in that after the suction of the phagolysate in a sterile container, it is purified by centrifugation for 30-60 minutes at 3000-6000 rpm and sterilizing filtration of the supernatant through a filter with a pore diameter of 0.2-0, 45 microns. 4. The method according to p. 1, characterized in that after suction of the phagolysate in a sterile container, it is purified by adding chloroform to it, keeping it for 15-45 minutes with continuous shuttling, centrifuging for 30-60 minutes at 3000-6000 rpm and sterilizing filtration of the supernatant through a filter with a pore diameter of 0.2-0.45 microns. 5. The method according to p. 1, characterized in that after the suction of the phagolysate in a sterile container, it is purified by filtration through a filter with a pore diameter of 0.45-1.0 μm and sterilizing filtration of the obtained filtrate through a filter with a pore diameter of 0.2-0, 45 microns. 6. The method according to any one of paragraphs. 1-5, characterized in that after sterilizing filtration pass the obtained filtrate through a column containing an agent affinity for endotoxin. 7. The method according to any one of paragraphs. 1-5, characterized in that before sterilizing filtration mix liquids containing bacteriophages, and then sterilize the mixture of liquids by filtration through a filter with a pore diameter of 0.2-0.45 microns. 8. The method according to p. 7, characterized in that after sterilizing filtration pass the obtained filtrate through a column containing an agent affinity for endotoxin. 9. The method according to p. 1, characterized in that as a vessel for cultivation using a glass microbiological mattress.