RU2142287C1 - Strains of bacteria bacillus subtilis and bacillus licheni-formis used as components of preparation against viral and bacterial infections and preparation based on these strains - Google Patents

Abstract

FIELD: microbiology, medicine, veterinary science. SUBSTANCE: strains of bacteria Bacillus subtilis VKPM B-7092, VKPM B-7048 and the strain of bacterium Bacillus licheniformis VKPM B-7038 are used as components of the preparation against viral and bacterial infections. The preparation has a mixture of the indicated strains biomass in the spore form at titer 3 x 10¹⁰ spores/g, not less. The proposed complex antibacterial preparation has also starch and sugar taken at the definite ratio of components. EFFECT: broadened spectrum of an antiviral and an antibacterial activity of the preparation. 4 cl, 8 tbl

Description

 The invention relates to biotechnology, namely genetic engineering, and can be used in medicine and veterinary medicine to obtain drugs against viral and bacterial infections.

 However, the known natural strain does not have antiviral activity. In addition, the strain has insufficient antagonistic activity, which is experimentally confirmed, for example, in relation to such test cultures as Pseudomonas vulgaris and Pseudomonas fluorescens. In this regard, preparations made on the basis of this strain will also have low antagonistic activity and not have antiviral activity.

 A known bacterial strain Bacillus licheniformis N 31 (Academician DZ Zabolotny Institute of Microbiology and Virology, Academy of Sciences of Ukraine), used as one of the components of the biosporin preparation [2], intended for the treatment of purulent-septic postpartum diseases.

 However, this strain does not have antiviral activity.

Known bacterial preparation based on a strain of Bacillus subtilis C-3102 [4]. The drug contains the indicated bacterial culture in dry form, as well as nutritional supplements for pharmaceutical or veterinary preparations. The technology for producing the drug includes suspension cultivation of a bacterial strain in a nutrient medium containing a carbon source, a nitrogen source, inorganic substances, vitamins, amino acids at a pH of 6-8 and a temperature of 35-40 ^o C for 0.5 to 7 days. The bacterial culture is separated from the nutrient medium, washed and concentrated. The resulting product is dried and mixed with filler additives.

 The disadvantage of this drug is that it does not have antiviral activity. In addition, the drug has a complex production technology, because it includes operations to wash bacterial cells from the components of the nutrient medium, and then concentrate the bacteria by centrifugation.

 The closest analogue of the proposed drug (prototype) is a bacterial therapeutic and prophylactic drug in liquid form, including the biomass of Bac bacteria. subtilis 5/6, the biomass of bacteria Bac. subtilis 2/10 and the biomass of bacteria Bac, licheniformis 31. A mixture of these components of the drug is a microbial association with a volume ratio of 1: 1: 1 (the content of each component of the drug is 100-150 billion microbial cells in 1 ml of saline). The specified liquid form of the drug is used to treat purulent-septic postpartum diseases [5]. The drug has a wide range of antagonistic activity.

 The disadvantage of this drug is that it has a liquid form, which is not stored for a long time and is not convenient to use, and also does not have antiviral activity.

 The objective of the invention is to obtain bacterial strains of Bacillus subtilis, Bacillus licheniformis and based on them the creation of a comprehensive bacterial preparation with a wider spectrum of antiviral and antibacterial activity.

To solve this problem, it is proposed:
- a bacterial strain Bacillus subtilis IC-9, deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), GNII Genetika under the number VKPM B-7048 dated 05/31/95. The strain was obtained from a natural isolate by selection. The strain was selected for inhibition of bacterial and fungal growth. With the best options, experiments were carried out to suppress growth: trichophytes, microsporiums, epidermophytons, candidomycoses and other fungi.

 - a bacterial strain Bacillus licheniformis IC-1, deposited in the All-Russian Collection of Industrial Microorganisms (VKPM), GNII Genetika under the number VKPM I-7038 dated 05/30/95. The strain was obtained from a natural isolate by selection. The strain was selected for inhibition of bacterial and fungal growth. With the best options, experiments were carried out to suppress the growth of pathogenic strains: staphylococcus, streptococcus, salmonella, trichophytes, microsporiums, epidermophytons, candidomycoses, etc.

 - the bacterial strain Bacillus subtilis IC-16 (pBMB5) obtained by transforming the B. subtilis strain VKPM B-7048 (IC-9) with the recombinant plasmid pBMB 5 DNA containing the α-2 interferon gene. The resulting strain IC-16 was deposited in the All-Russian Collection of Industrial Microorganisms (VKPM) GNII Genetika under the number VKPM B-7092. The plasmid pBMB 5 is able to replicate in bacilli and contains the human α-2 interferon gene, a promoter for the expression of this gene, a ribosome landing site (SD site) and the signaling peptide of α amylase B. amyloliquefaciens for the secretion of interferon into the culture medium, as well as bearing resistance to kanamycin.

In addition, this problem is solved in that in the preparation against viral and bacterial infections, including a mixture of biomass strains of Bacillus subtilis and Bacillus licheniformis in equal proportions, according to the invention, it additionally contains starch and sugar, and as a biomass of bacterial strains Bacillus subtilis and Bacillus licheniformis use the biomass of bacterial strains Bacillus subtilis VKPM B-7092, VKPM B-7048 and Bacillus licheniformis VKPM B-7038 in spore form with a titer of at least 3 • 10 ¹⁰ spores / g in the following quantitative ratio of components (wt.%):
A mixture of spores of bacteria Bacillus subtilis and Bacillus licheniformis in equal proportions - 0.1 - 1.0
Starch - 1.0 - 3.0
Sugar - Else
A high titer of bacterial spores (10 ¹⁰ spores / g or more) is ensured by creating the same conditions for spore formation for each bacterial cell.

 The immobilization of bacterial spores on starch provides them with additional mechanical protection, prevents the adhesion of bacterial spores and ensures a more even distribution in the mass of the filler.

 When using the drug, starch helps accelerate the germination of bacterial spores, which increases its effectiveness.

 Sugar is not only a sorbent-filler, but also has a stabilizing and preserving effect on the preparation, creates milder conditions for storing bacterial spores.

 In addition, the proposed method greatly simplifies the technology of obtaining the drug both at the stages of cultivation (the technology does not use expensive enzymes, cleaning and concentration of the drug is not required), and at the stages of obtaining its finished form (removal or redistribution of moisture combined with obtaining the finished form of the drug) .

Cultural, morphological and biochemical properties of the Bac strain. subtilis VKPM B-7048
The strain VKPM B-7048 refers to aerobes, a gram-positive bacterium. When grown on medium, LA gives a colony shape similar to chamomile. The colonies are whitish. The strain has the appearance of sticks. The size of the cells of a one-day agar culture is (2-4) • (0.5-0.8) microns. Disputes form. Capsules do not form. Gram stains positively. The strain multiplies at 15-50 ^o C, optimal growth at a temperature of 36-37 ^o C.

 Well form endospores on potato agar. Endospores are ellipsoidal, located centrally and do not go beyond the size of sporangium. Near-peroral inclusions were not detected.

After 48 hours of growth at +37 ^o C on wort, the culture has opaque matte colonies with a scalloped margin. It is well removed by a loop from an agar surface.

 FH medium: minimum 4; maximum 8; optimal 6-7.

The strain multiplies at 37 ^o C on environments MPV and MPA.

 Biochemical properties. Causes hydrolysis of starch, reduction of nitrates. Breaks down glucose, sucrose, mannitol maltose and lactose.

 The strain is not phytopathogenic.

 Strain VKPM B-7048 produces a broad-spectrum antibiotic that inhibits the growth of fungi, staphylococci, streptococci and Pseudomonas aeruginosa.

 The culture does not grow under anaerobic conditions and at 10% NaCl, does not form gas from NO under anaerobic conditions.

With prolonged storage of the strain, the substance is lyophilized. For the reproduction of bacteria using meat peptone agar (MPA), and the cultivation is carried out at a temperature of 37 ^o C.

Cultural, morphological and biochemical properties of the Bac strain. licheniformis VKPM B-7038
Strain VKPM B-7038 relates to aerobes; gram-positive bacillus capable of forming spores. The sizes of vegetative cells (0.5-0.8) microns and (1.5-3) microns, sometimes reach up to 5 microns. The spores are elliptical, mainly located centrally, no more than one in the sporangia cell, without distinct bloating of the sporangia.

Vegetative cells of B. licheniformis VKPM B-7038 grow well on rich nutrient media (meat-peptone broth - MPB and meat-peptone agar - MPA) at a temperature of (37-40) ^o C. There is no growth in the presence of 0.02% azide, but there is an increase in Saburo agar, with 7% NaCl and with the addition of 0.001% lysozyme.

 Weak growth is observed in anaerobic agar.

On a meat and peptone broth, pH (7.3 ± 0.1) after 48 hours of incubation at a temperature of (37 ± 1) ^o C forms a turbidity of the medium, and on the surface a dry film; on Hottinger’s meat and peptone agar, pH (7.3 ± 0.1) —weakly convex dry opaque whitish colonies, polymorphic, fine-grained with a diameter of (4-6) mm, growing into the nutrient medium.

To obtain spores, a culture grown on solid nutrient medium is transferred through a bacteriological loop into tubes or vials on potato or wheat agar, closed with cotton-gauze swabs and incubated at a temperature of 37 ^o C for (7-10) days at an angle of 45 ^o C agar up. After completion of sporulation, the culture is stored at 4 ^° C. in a refrigerator.

 Biochemical properties. Vegetative cells of B. licheniformis VKPM B-7038 hydrolyze casein and starch, are catalase-positive. The Voges-Proskauer reaction is positive.

 The strain is not pathogenic for plants, animals and humans.

 Strain VKPM B-7038 produces a broad-spectrum antibiotic that inhibits the growth of fungi, staphylococci, streptococci and Pseudomonas aeruginosa.

 During prolonged storage of the strain, the substance is lyophilized.

The technology of obtaining the recombinant strain of B. subilis VKPM B-7092 (IC-16) and the characteristic of its properties
To transmit genetic information, the B. subtilis protoplast transformation method described by Chang and Cohen is used [6]. The technique is as follows. 50 ml of LB medium (Luria broth) in a 500 ml flask was inoculated with 2.5 ml of B. subtilis VKPM B-7048 (IC-9) overnight culture and grown under intensive aeration to D ₆₀₀ = 0.4 ^o / ml. The cells are then pelleted by centrifugation in a JA-20 rotor at 6000 rpm for 10 minutes, the supernatant is removed, and the cells are resuspended in 5 ml of SMMP medium. An SMMP medium is prepared by mixing equal volumes of 2xSMM and LBP. Composition of 2xSMM buffer: 0.5 M sucrose, 0.02 M Na maleate, 0.02 M MgCl ₂ , pH 6.5. The medium is autoclaved at 0.7 atm. The composition of the LBP medium (per 1 l): 20 g of tryptone ("Difco"), 10 g of NaCl, 10 g of yeast extract ("Difco"), 80 g of polyvinylpyrrolidone ("Fluka"). The medium is autoclaved at 0.7 atm.

 0.5 ml of a solution containing 20 mg / ml lysozyme was added to the resuspended cells (the lysozyme solution in SMMP was prepared shortly before use and sterilized by filtration through 0.2 nm Millipore filters).

Cells are incubated at 37 ^o C with gentle stirring. Aliquots are taken at specific intervals and examined under a phase contrast microscope to determine the number of protoplasts. When at least 99% of the cells turn into protoplasts (1-1.5 hours), they are pelleted in the JA-20 rotor for 15 min at 5000 rpm, washed 1 time by resuspension in SMMP and centrifuged again under the same conditions .

Next, the protoplasts are resuspended in 1/15 of the starting culture volume. those. in 3.3 ml of SMMP, add 5-10 μg of plasmid DNA and 1.5 ml of a 40% solution of polyethylene glycol, dissolve 50 ml of 2xSMM, bring to 100 ml with water, autoclave at 0.7 atm, mix gently and leave for 2 min at room temperature. Then 5 ml of SMMP medium is added to dilute the polyethylene glycol, mixed and centrifuged in a JA-20 rotor at 5000 rpm for 10 minutes. Protoplasts are resuspended in 1 ml of SMMP medium and incubated at 37 ^° C. with gentle stirring for 1.5 hours. Aliquots of 0.1 ml are seeded into plates with DM3 medium containing 1 mg / ml kanamycin for the regeneration of protoplasts. The composition of the DM3 medium (per 1 l): 200 ml of 4% agar ("Difco"), 500 ml of 1 M Na succinate, pH 7.3, 100 ml of 5% casamino acids (Difco), 50 ml of 10% yeast extract ("Difco"), 100 ml of 3.5% K ₂ HPO ₄ and 1.5% KH ₂ PO ₄ , 25 ml of 20% glucose, 20 ml of 1 M MgCl ₂ . After autoclaving at 0.7 atm, 5 ml of 2% BSA is added to the medium.

 The procedure described above is used to control the transformation of B.subtilis protoplasts, but the same volume of TE buffer (10 mM HCl), pH 8.0, 1 mM ED-TA) or water is added instead of plasmid DNA. To control the intactness and viability of protoplasts, one aliquot is seeded on a medium that does not contain kanamycin).

 As a result of the procedures performed on the medium with kanamycin, 7 clones were obtained (after transformation of B. subtilis plasmid DNA pBMB 5). Control plates containing protoplasts after adding TE buffer to them were empty. On a plate with medium without kanamycin, the protoplasts grew solid lawn.

 Kanamycin-resistant protoplasts were streaked onto LA Petri dishes (Luria agar broth) containing 50 μg / ml kanamycin. For control, LA medium with 50 μg / ml ampicillin and LA with 10 μg / ml tetracycline are used. All 7 protoplasts showed good growth on medium with kanamycin, weak growth on medium with ampicillin and no growth on medium with tetracycline. The original B. subtilis strain does not grow on medium with kanamycin or tetracycline and has weak growth on medium with ampicillin.

To describe the colony morphology of the obtained 7 protoplasts, a suspension of these cultures in physiological saline (0.85% NaCl) is plated on JA medium and grown at 37 ^° C. for 18 hours. As a control, the original B. subtilis IC-9 strain (VKPM-7048) is used.

 5 out of 7 protoplast colonies have the same morphology as the original B. subtilis IC-9 strain. The diameter of the colonies is 0.3-0.5 mm. The colonies are rough, resembling chamomile, i.e. a recess in the middle, and then a dense roller bordering the “core”, and more transparent edges, slightly wavy.

When stained according to Orzeszko [7] and microscopy of smears of cultures grown on JA medium overnight at 37 ^° C and kept for 3 hours in a refrigerator, oblong vegetative sticks and oval spores were found. All 7 protoplasts and the original B. subtilis strain have the same picture.

 All protoplasts exhibit stability of plasmid maintenance in cells, i.e., when stored on JA medium with 50 μg / mg kanamycin and subsequent subcultures on a similar medium, no sign of resistance to kanamycin was observed.

 To isolate the plasmid from clones after transformation of the initial strain, the Birnboim and Doly method was used [8]. Using electrophoresis in 0.8% agarose gel and subsequent staining with ethidium bromide, the presence of the plasmid in the transformed clones was shown.

 Strain B. subtilis VKPM B-7092 is characterized by the following properties.

Cultural - morphological properties. B. subtilis belongs to aerobes. When grown on medium, LA gives a colony shape similar to chamomile. The colonies are whitish. Bacteria look like sticks. The size of the cells of a one-day agar culture is (2-4) • (0.5-0.8) microns. Disputes form. Capsules do not form. Gram stains positively. The strain multiplies at 15-50 ^o C, optimal growth at a temperature of 36-37 ^o C.

 Biochemical properties. Causes hydrolysis of starch, reduction of nitrates. Breaks down glucose, sucrose, mannitol, maltose and lactose.

 The strain is resistant to kanamycin and is not pathogenic.

 Strain VKPM B-7092 synthesizes α2-interferon and a broad-spectrum antibiotic that inhibits the growth of fungi, staphylococci, streptococci and Pseudomonas aeruginosa.

During prolonged storage of the strain, the substance is lyophilized. For the reproduction of bacteria using meat peptone agar (MPA), and the cultivation is carried out at a temperature of 37 ^o C.

Example 1. The study of the pathogenicity of B. subtilis VKPM-7048
The study was conducted in the laboratory of antibacterial agents and microbiological toxicology of the All-Russian Scientific Center for the Safety of Biologically Active Substances (VSC BAS).

 The pathogenicity of the microorganism was assessed by the survival of infected animals, by their appearance and general behavior, bacterial seeding from blood and organs at various times after infection, as well as by a macroscopic picture of the internal organs when opening the animals at the end of the observation period.

 Two types of laboratory animals were used in the work. The experiments were performed on 12 nonlinear white mice (18-20 g) and 4 white rats (180-200 g).

The studied B. subtilis strain was grown on Hottinger agar at 32 ^o C for a day. The infectious dose for animals was 10 microbial bodies per animal. The microbial suspension was administered intraperitoneally in 0.5 ml of isotonic sodium chloride solution. Animals were observed for 30 days.

 Microorganisms were seeded from blood and organs on Hottinger agar in Petri dishes. In mice, a microbiological analysis of blood, liver and spleen was performed three times during the observation period - 3, 7 and 14 days after the introduction of the studied strain, in rats - once after 14 days. In all cases, 2 animals of each species were taken.

 At the end of the observation period (30 days), 2 animals of each species were killed with ethyl ether, opened and a macroscopic evaluation of the internal organs: heart, lungs, stomach, liver, kidneys and spleen was performed.

 As a result of the studies established the following. No animal deaths were recorded during the experiment. No changes in their appearance and behavior were noted. A macroscopic evaluation of the internal organs on the 30th day of observation of pathological changes was not detected.

 Microbiological analysis did not show the seeding of the microorganism from the blood and internal organs of animals at all stages of the study.

 Based on the absence of animal death and any changes in their appearance and general behavior during a 30-day observation, negative results of a microbiological analysis of blood, liver and spleen, normal picture of the internal organs of animals during autopsy at the end of the observation period, it was concluded that Bacillus subtilis strain IC-9 (VKPM B-7048) is non-pathogenic.

Example 2. The study of the pathogenicity of the strain of Bacillus licheniformis VKPM-7038
The studied strain of B. licheniformis was grown on Hottinger agar at 35 ^o C for a day.

Studies were conducted on white mice. The infectious dose for animals was 10 ¹⁰ microbial bodies per animal. The microbial suspension was administered intraperitoneally in 0.5 ml of isotonic sodium chloride solution. Animals were observed for 30 days.

 Microorganisms were seeded from blood and organs on Hottinger agar in Petri dishes. In mice, a microbiological analysis of blood, liver, and spleen was performed three times during the observation period — 3, 7, and 14 days after administration of the studied strain, and in rats — once every 14 days. In all cases, 2 animals of each species were taken.

 At the end of the observation period (30 days), the animals were sacrificed with ethyl ether, opened and a macroscopic evaluation of the internal organs: heart, lungs, stomach, liver, kidneys and spleen was performed.

 As a result of the studies established the following. No animal deaths were recorded during the experiment. No changes in their appearance and behavior were noted. A macroscopic evaluation of the internal organs on the 30th day of observation of pathological changes was not detected.

 Microbiological analysis did not show inoculation of the microorganism from the blood and internal organs of animals after 7 days of the study.

 Based on the absence of animal death and any changes in their appearance and general behavior during a 30-day observation, negative results of a microbiological analysis of blood, liver and spleen, normal picture of the internal organs of animals during autopsy at the end of the observation period, it was concluded that Bacillus licheniformis strain VKPM B-7038 is non-pathogenic.

 Data on the non-pathogenicity of the Bac strain. licheniformis VKPM B-7038 are presented in table 1.

 Analysis of table 1 shows that the proposed strain is harmless to animals.

Example 3. The study of the pathogenicity of the bacterial strain Bacillus subtilis VKPM-7092
The study was conducted in the laboratory of antibacterial agents and microbiological toxicology of the All-Russian Scientific Center for the Safety of Biologically Active Substances (VSC BAS).

 The pathogenicity of the microorganism was assessed by the survival of infected animals, by their appearance and general behavior, bacterial seeding from blood and organs at various times after infection, as well as by a macroscopic picture of the internal organs when opening the animals at the end of the observation period.

 Two types of laboratory animals were used in the work. The experiments were performed on 12 nonlinear white mice (18-20 g) and 4 white rats (180-200 g).

The studied B. subtilis strain was grown on Hottinger agar at 32 ^o C for a day. The infectious dose for animals was 10 microbial bodies per animal. The microbial suspension was administered intraperitoneally in 0.5 ml of isotonic sodium chloride solution. Animals were observed for 30 days.

 Microorganisms were seeded from blood and organs on Hottinger agar in Petri dishes. In mice, a microbiological analysis of blood, liver, and spleen was performed three times during the observation period — 3, 7, and 14 days after administration of the studied strain, and in rats — once every 14 days. In all cases, 2 animals of each species were taken.

 At the end of the observation period (30 days), 2 animals of each species were killed with ethyl ether, opened and a macroscopic evaluation of the internal organs: heart, lungs, stomach, liver, kidneys and spleen was performed.

 As a result of the studies established the following. No animal deaths were recorded during the experiment. No changes in their appearance and behavior were noted. A macroscopic evaluation of the internal organs on the 30th day of observation of pathological changes was not detected.

 Microbiological analysis did not show the seeding of the microorganism from the blood and internal organs of animals at all stages of the study.

 Based on the absence of animal death and any changes in their appearance and general behavior during a 30-day observation, negative results of a microbiological analysis of blood, liver and spleen, normal picture of the internal organs of animals during autopsy at the end of the observation period, it was concluded that Bacillus subtilis strain IC-16 (VKPM B-7092) is non-pathogenic.

Example 4. The study of the antagonistic activity of strains of B. subitilis VKPM B-7048, VKPM B-7092 and B. licheniformis VKPM B-7038
The selectively obtained B. subtilis strain VKPM B-7048 is characterized by high antagonistic activity against pathogenic and conditionally pathogenic microorganisms.

 Antagonistic activity against test cultures is checked by the method of delayed antagonism. Shigella sonnei, Salmonella typhimurium are used as test cultures. S. stenly, S. reading, S. derby, Staphylococous aureus, Pseudomonas aeruginose, Proteus vulgaris, Klebsiella pneumoniae, Candida albicans, C. tropicalis, etc. The test microbes used must meet the following requirements: be in S-form, have typical morphological and enzymatic properties.

 Used strains of test microbes gave a positive keratoconjunctival test in guinea pigs.

 Staphylococcus test strains formed a 3-5 mm hemolysis zone around their colonies after 18-20 hours of growth on blood agar, coagulated rabbit blood plasma for 2 hours, caused necrosis for 48-72 hours with 200 million intradermal administration to the rabbit microbial cells of a daily culture in 0.2 ml of physiological saline.

 The study of the antagonistic activity of B. subtilis strains VKPM B-7048, VKPM B-7092 and B. licheniformis VKPM B-7038 is carried out on a dense Gause medium N 2.

The formulation of a dense medium Gause N 2 1 l:
1. Hottinger broth (700 mg% total nitrogen) - 30 ml
2. Peptone - 5 g
3. Sodium chloride - 5 g
4. Glucose - 10 g
5. Microbiological agar - 15 g
6. Distilled water - up to a total volume of 1 liter.

After mixing the components, the medium is heated until the agar is completely dissolved and filtered through a cotton-gauze filter into 0.3-0.4 l flasks. The flasks with the medium are sterilized in an autoclave at a pressure of 0.1 MPa for 15 minutes. After cooling to a temperature of (45 ± 2) ^o C, the medium is poured into sterile Petri dishes. Petri dishes with the medium are placed in a thermostat and incubated at a temperature of (37 ± 1) ^o C for (24 ± 2) hours to verify sterility.

Cultures of Shigella sonnei, Salmonella typhimurium, Staphylococcus aureus, Candida albicans and other above test cultures were grown on Petri dishes for (18 + 2) hours on meat peptone agar (MPA). 1 ml of physiological saline solution is poured into sterile tubes and suspensions of test strains with a concentration of (5.0 ± 0.5) • 10 ⁸ cells / ml are prepared.

 For testing, 4 samples of 2 g are taken from each series.

Then, 0.5 g of the preparation based on the B. subtilis strain 7048 is diluted in 1 ml of physiological saline. The resulting suspension is streaked using a microbiological loop along the diameter of the Petri dish with a dense Gause medium N 2. Crops are incubated in an incubator at (37 ± 1) ^o C for (72 ± 2) hours. Then the test microbe seed is seeded to the grown culture by the method of perpendicular strokes.

Analysis of the results is carried out after 18 hours of incubation at (37 ± 1) ^o C according to the size of the zones of lack of growth of test cultures. The growth control of test cultures is controlled by their parallel plating on plates with the same dense Gauze N 2 medium without the studied culture association.

The B. subtilis strains VKPM B-7048 and VKPM B-7092 are characterized by the following indicators of growth inhibition zones of the test culture (mm):
Shigella sonnei - 17-22
Salmonella typhimarium - 21-25
S. stenly - 13-17
S. reading - 14-18
S. derby - 15-18
Staphylococcus aureus - 22-27
Pseudomonas fluoresocens - 8-10
Pseudomonas vulgaris - 10-12
Pseudomonas aeruginose - 7-9
Proteus vulgaris - 12-14
Klebsiella pheumoniae - 13-15
Candida albicans - 25-29
C. tropicalis - 22-26
Strain B. licheniformis 7038 is characterized by the following indicators of growth inhibition zones of the test culture (mm):
Shigella sonnei - 22-27
Salmonella typhimurim - 26-30
S. stenly - 18-22
S. reading - 19-23
S. derby - 20-23
Stapgylococcus aureus - 27-33
Pseudomonas aeruginose - 12-15
Proteus vulgaris - 17-19
Klebsiella pneumoniae - 18-20
Candida albicans - 30-34
C. tropicalis - 27-31
Thus, the studied bacterial strains have high antagonistic activity against a wide range of pathogenic and conditionally pathogenic microorganisms.

 In addition, the antibiotic activity of production strains of R. subtilis VKPM B-7048, VKPM B-7092 and B. licheniformis VKPM B-7038 with respect to each other as components of complex preparations was studied. In this case, we can conclude that the antagonism of B. subtilis and B. licheniformis cells is not a significant obstacle to their joint use in drugs.

Example 5. The study of the antiviral activity of the strain B. subtilis VKPM I-7092
To study the antiviral activity, a bacterial strain is cultivated on a solid nutrient medium of the following composition:
Agar-agar - 20 g / l
Peptone - 12 g / l
Yeast extract - 5 g / l
NaCl - 5 g / l
Water - Up to 1 liter
Kanamycin - 50 g / l
25 g of bacteria with a titer of about 10 ⁸ cells / ml are required per 1 liter of agar medium. The nutrient medium is formed in the container in the form of a layer on which a semipermeable membrane, for example a cellophane film, is laid. A seed dose of the bacterial strain is applied on top of the film. Cultivation is carried out in a thermostat at a temperature of 34-37 ^o C for 20-24 hours. Next, the container is transferred to a thermostat with a temperature of + (6-10) ^o C and maintained at this temperature for 1 day until the formation of bacterial spores. With this cultivation method, a spore titer of 3 x 10 ¹⁰ spores / g can be obtained. Bacterial spores are removed with a glass spatula from the surface of the cellophane film and collected in a sterile container. These bacterial spores are pure biomass concentrate that is tested for antiviral activity.

 The antiviral activity of the VKPM B-7092 strain is determined by the inhibition of the cytopathic effect of the vesicular stomatitis virus (BBC) on the L-68 cell culture.

The Air Force is pre-passaged on chicken embryos according to TU 42-14-99-77, at least 3 passages are performed at an infectious dose of 100-1000 TCD ₅₀ / 0.1 ml. Infectious activity of the material should be 1 • 10 ⁶ - 1 • 10 ⁷ TCD ₅₀ / ml. A monolayer of L-68 cells is grown on a mattress with a capacity of 1000 ml in the presence of a growth medium (Migla MEM medium with a double set of ingredients - 90% FS 42-7BC-90, serum of cow fruits FS 42-7BC-90-10%) s antibiotics (kanamycin 100 thousand units / ml, gentamicin 16 units / ml), after which the cells are removed from the glass, suspended in 40 ml of growth medium and their number is counted in the Goryaev counting chamber. After this, the cell suspension is diluted with growth medium so that 1.0 ml contains 200 thousand cells / ml. 0.1 ml of cell suspension is added to the wells of the microplates and after 2-3 days of incubation at 37 ^° C, a monolayer culture is obtained.

After the monolayer is formed, the growth medium is aspirated, 20 μl of a sample of a sterile filtrate of cells of VKPM strain B-7092 obtained as described above and 180 μl of support medium are introduced into the first well; after stirring gently, 100 μl from the first well is transferred to the second well, mixed with 100 μl of support medium, etc. Cells were incubated for 24 hours at 37 ^o C and after that 100 tissue cytopathic doses, causing damage in half of the samples, of the BBC virus were added to each well and kept at 37 ^o C for 24-48 hours. After 1 day, the first count was carried out, after 2 days - second. The amount of antiviral activity in the test samples is determined by comparison with the antiviral activity of the reference drug, which is used as a reaferon drug, and expressed in international units (ME).

All the studied samples possess antiviral activity of 3 • 10 ⁵ -5 • 10 ⁵ ME / l, while in the samples obtained from the plasmid-free B.subtilis strain IC-9, no antiviral activity was detected.

 Example 6. Determination of the stability of the recombinant plasmid rBMB 5 carrying the synthetic gene α-2 of human interferon in the bacterial cells of B. subtilis VKPM B-7092.

 B. subtilis cells are grown in 2.LB medium with starch until the late stationary phase, then transferred to fresh medium and grown again until the late stationary phase, etc., a total of 10 transfers. After each reseeding, seeding is performed on MPA without an antibiotic. The grown isolated colonies are transferred to plates with MPA with kanamycin and without antibiotic on a nitrocellulose filter. After growing, the number of colonies grown on MPA with an antibiotic and without an antibiotic is calculated and compared. In this way, the number of clones that have lost the recombinant plasmid after each reseeding is determined. The absence of the plasmid is confirmed by plasmid DNA isolation.

 The safety of the α-2 type human interferon gene in the recombinant plasmid pMBB 5 after reseeding is confirmed by the hybridization method. Filters with colony imprints after passages are inhibited with a labeled fragment containing the α-2 type interferon gene isolated from rBMB 5 plasmid. All clones that have not lost recombinant plasmids after passages are hybridized with the labeled fragment containing the interferon gene, which indicates the preservation of the interferon gene type α-2 as part of the plasmid rBMB 5. In addition, plasmid DNA is isolated from randomly selected clones and analyzed by restriction endonucleases.

 It was found that plasmid DNA is preserved and inherited for 10 passages on MPA, which indicates its stability.

Example 7. The method of cultivation of bacteria of the genus Bacillus to obtain bacterial biomass
To obtain biomass of bacterial spores, B.licheniformis VKPM-7038, B.subtilis VKPM B-7092, B.subtilis VKPM B-7048, and B.subtilis VKPM B-7048 strains of bacteria are separately cultivated on a solid nutrient medium of the following composition:
Agar-agar - 20 g / l
Peptone - 12 g / l
Yeast extract - 5 g / l
NaCl - 5 g / l
Water - Up to 1 liter
For 1 liter of agar medium, 25-30 ml of the same species of bacteria with a titer of (10 ⁶ -10) ⁸ cells / ml are required. The nutrient medium is formed in containers in the form of layers on which the inoculative doses of each specified type of bacteria of the genus Bacillus are applied. The cultivation of each type of bacteria is carried out separately in a thermostat at a temperature of + (34-37) ^o C ^o for 20-24 hours. Next, the containers are transferred to a thermostat with a temperature of + (6-10) ^o C and maintained at this temperature for 1-2 days until the formation of bacterial spores. With this cultivation method, a spore titer of (3-10) • 10 ¹⁰ spores / g can be obtained.

Example 8. Obtaining the finished product based on the microbial association of the genus Bacillus
First, a mixture of sugar with starch in a ratio of 1: 1 is prepared separately for the biomass of each type of bacteria. Then, to 5 parts of the obtained filler mixture, add 1 part of the bacterial spore biomass of the VKPM B-7092 strain or VKPM B-7048 strain and mix. In this case, the bacterial spores are immobilized on starch particles. Next, sugar is introduced into the resulting mixture at a ratio of biomass of spores of bacteria and sugar of not more than 1:50. The result is the components of the drug in dry form in the following ratio of components (wt.%):
1. component A
Bac. subtilis VKPM B-7092 with a titer of at least 3 • 10 ¹⁰ spores / g - 0.1-1.0
Starch - 1.0-3.0
Sugar - Else
2. component B
Spores of bacteria Bac.subtilis VKPM B-7048 with a titer of at least 3 • 10 ¹⁰ spores / g - 0.1-1.0
Starch - 1.0-3.0
Sugar - Else
3. component B
Bac. subtilis VKPM B-7092 with a titer of at least 3 • 10 ¹⁰ spores / g - 0.1-1.0
Starch - 1.0-3.0
Sugar - Else
Next, the components A, B, and C of the drug in dry form are mixed in a ratio of 1: 1: 1 and get the finished form of the drug.

The residual moisture content of the drug is not more than 2-5%
Example 9. Data on the storage of the dry form of the drug based on the association of spores of bacteria of the genus Bacillus
To establish the shelf life and storage conditions of the preparations, relevant studies were conducted.

From table 2 it follows that within 12 months of storage of the dry form of the drug at a temperature of +24 ^o C, the titer practically did not change, which confirms the high thermal stability of the preparations in spore form, which provides a significant simplification of the technology of their use in agriculture and medicine.

Example 10. The use of a dry form of the drug based on the association of spores of bacteria of the genus Bacillus
The drug has both antibacterial and antiviral activity due to the high antagonistic activity of the microbial association of the supports of bacteria of the genus Bacillus and the action of alpha-2-interferon, and also stimulates cellular and humoral factors of immunity and is able to increase nonspecific resistance of the body.

 The drug is used orally for all types of mammals for the treatment and prevention of colibacteriosis, dysfunctions and dysbacterioses of various nature, plague, parainfluenza, influenza, dyspepsia, salmonella, infectious rhinotracheitis in cattle, viral diarrhea, rotavirus enteritis, dysentery, staphylococcus dermatitis, staphylococcus dermatitis as well as for the prevention of immunodeficiency conditions.

 For treatment, the drug is administered orally at a dose of 50 mg / kg weight twice a day with an interval of 12 hours or at a dose of 75 mg / kg of weight once a day until the body recovers.

 The bacterial association used in the preparation is highly resistant to the digestive juices and enzymes of the gastrointestinal tract of mammals and the ability to rapidly colonize the stomach and intestines. When taken orally with water or food, bacteria multiply in the gastrointestinal tract for 3-6 days, then they are completely eliminated from the body without causing negative consequences even with an overdose.

 Propagating, bacteria with their proteases lyse all proteins unusual for the mammalian organism, denatured proteins and nucleoproteins. In this case, bacterial toxins, elements of tumor neoplasms and other defective cells are destroyed. Healthy cells remain intact due to the presence of anti-enzyme inhibitors in them. Bacteria of the B. subtilis strain VKPM B-7092 in the course of their life produce interferon. Through the walls of the intestine, interferon enters the bloodstream. At the same time, the phagocytic activity of blood leukocytes increases, the body's immune status and resistance to various types of viral and other diseases increase. The regenerative processes of body tissues are stabilized. Metabolism is normalized. A humoral immune response is stimulated, and antigen-reactive T-lymphocytes activate the functions of peritoneal macrophages.

Example 11. The study of the effectiveness of treatment of plague carnivores
For this, from the affected dogs diagnosed with the intestinal form of carnivore plague in the initial stage, 1 control and 4 experimental groups of 5 animals each were formed (Table 3).

The scheme of experiments in the treatment of plague carnivores
Treatment by the proposed method was carried out for 5-6 days. After the disappearance of clinical signs to animals, the proposed drug was administered orally for 3-5 days once after two days in order to prevent possible relapses. In the control group, hyperimmune serum, γ-globulins, antibiotics, sulfonamides, vitamins, and heart were used for treatment.

The results of treatment of plague
When using the proposed method, the effectiveness of the treatment of intestinal forms of dog plague increases. Moreover, there is a direct relationship between the dose of the proposed drug and the severity of its therapeutic effect. So, when prescribing the drug at a dose of 50 mg / kg weight after 12 hours and at a dose of 75 mg / kg once a day, optimal results were obtained. When treating animal patients with plague using the proposed drug in the above dosages, a 100% positive effect was achieved without the use of traditional serums and immunoglobulins. Improvement in the course of the disease is already observed for 2-3 days and in the future the disease proceeds in a milder form in comparison with analogues from the control group.

 Under the influence of treatment by the proposed method, a functional reorganization of the body of a positive nature occurs. The functions of vital organs and systems are stimulated. Redox processes increase and metabolism is activated. An increase in the number of lymphocytes and white blood cells may indicate an increase in the protective functions of the dog's body.

 In the control group, when using complex therapy, the disease was severe and the duration of treatment ranged from (10 -12) days.

 Thus, the proposed method is effective in the treatment of intestinal forms of dog plague at the beginning of the disease. Optimum results were obtained when using the drug at a dose of 50 mg / kg of weight after 12 hours and at a dose of 75 mg / kg of weight once a day.

Example 12. The study of the effectiveness of treatment of parvovirus enteritis
To study the effectiveness of the proposed method in the treatment of dogs parvovirus enteritis, 1 control group and 4 experimental groups of 5 dogs each with a diagnosis of parvovirus enteritis were also formed (Table 4).

 The duration of treatment was determined by the presence of symptoms of the disease. After the disappearance of clinical signs, taking into account the peculiarities of the course of the disease, the sick animals continued to conduct a preventive course at a dose of 50 mg / kg for 3-4 days, once every two days. Animals of all experimental groups were prescribed rehydration agents.

 For the treatment of animals of the control group, cardiac, antispasmodic and antiemetic drugs, antibiotics, rehydration agents and hyperimmune serum were used.

 The criteria for evaluating the effectiveness of the treatment course were the duration, nature and severity of the course of the disease, outcome, changes in the morphological parameters of the blood of animals. Blood in dogs was examined before treatment, during treatment (on day 5) and after recovery.

Parvovirus Enteritis Treatment Results
When parvovirus enteritis in dogs, the proposed method has a pronounced therapeutic effect. Optimal results were obtained when the drug was administered orally, in the initial period of the disease, in doses of 50 mg / kg of weight after 12 hours and at a dose of 75 mg / kg of weight once a day until the animals recover.

 The duration of treatment of parvovirus enteritis with the proposed method using the drug in doses of 50 mg / kg weight after 12 hours and at a dose of 75 mg / kg weight 1 time per day, depending on the severity of the disease, ranged from 2 to 5 days.

 Under the influence of the proposed drug, the nature of the manifestation and development of the pathological process changes. When prescribing the drug in the initial stage of the disease, intestinal inflammation was predominantly catarrhal and the dogs had no hemorrhagic discharge. One of the main clinical symptoms of parvovirus enteritis is a sharp inhibition of leukopoiesis. The proposed method eliminates the development of leukopenia. To a lesser extent, animals develop intoxication and dehydration phenomena.

 Under the influence of the proposed drug, the development of other viral infections of dogs (plague, hepatitis) and secondary bacterial infections, which are often observed in dogs that have had parvovirus enteritis, and, as a rule, are often fatal, is prevented.

 In animals of the control group, the disease progressed with moderate severity, mainly with hemorrhagic inflammation of the intestine and severe leukopenia. The duration of treatment of animals in the control group, depending on the severity of the disease ranged from 5 to 7 days.

Example 13. The study of the effectiveness of the prevention of piglets of the proposed method
Two sows with a litter at the age of 19 days were used in the experiment. Piglets of the experimental group were prescribed a drug at a dose of 50 mg / kg after 12 hours for 5 days. The drug was not administered to the piglets of the control group. There were 9 piglets in the experimental group, and 10 piglets in the control group.

 To determine the effectiveness of the preventive effect of the drug in the blood, the content of erythrocytes, leukocytes, hemoglobin, ROE, leukogram, subpopulations of T-lymphocytes, IgM and IgG was determined. Also considered indicators of the safety of piglets before weaning. In case of disease, the piglets were determined by the duration, nature of the course and outcome of the disease.

 In the study of blood of piglets before the introduction of the drug, no characteristic differences in the morphological and immunological parameters of the blood serum of animals of the experimental and control groups were recorded (tables 5, 6).

 The results of a blood test on the 10th day of the experiment indicate an increase in the number of red blood cells by 14.2% and the hemoglobin content by 10%. The content of leukocytes increased by 32.7%, segmented neutrophils by 18% and lymphocytes by 9% in comparison with analogues from the control group (Table 7).

 In both experimental and control piglets, quantitative changes in all subpopulations of T-lymphocytes, IgM and IgG occur in the direction of their increase, which is due to age-related specifics. However, in animals of the experimental group, these indicators were significantly higher than in analogues from the control group (table. 8).

Thus, the proposed drug stimulates the formation and output:
- poorly differentiated T-lymphocytes;
- functionally mature T-inductors-helpers and T-killers
- suppressors;
- T-active lymphocytes.

 Under the influence of the drug, the content of IgG in the blood increases. Therefore, the drug in the proposed method increases the natural resistance of the body, activating the T - and B-links of the immune system. The drug stimulates the factors of cellular immunity more intensively and to a lesser extent affects humoral factors. In the blood of experimental piglets, the content of T-killer suppressors significantly increases (by 32%), the activity of T-lymphocytes increases (by 51.7%), the content of post-thymic precursors of functionally mature cells increases (by 58.5%). The concentration of IgG in the blood increases by 20.8% compared with the control. The decrease in the content of IgM in the experimental group is due to the fact that they are likely to participate in antigenically dependent cellular cytotoxicity and are intensively fixed by killer cells and antigens, and therefore, their yield in the reaction of radial simple immunodiffusion is lower.

 Pigs in the experimental group were not sick, they grew and developed better. The safety of piglets in the experimental group was 100%, and in the control group - 83.3%. The average increase in live weight during the weaning period in the control group was (14.5 + 4.45) kg, in the experimental group (15.2 + 1.35) kg. Therefore, in the experimental group, the average increase in live weight is 4.83% higher than analogues from the control group. In the experimental group, the growth rate of piglets was uniform, with the exception of one piglet, the difference in growth was 1 kg, and in the control group - up to 8 kg.

 Animals tolerate the drug well; adverse pigment adverse events were not observed.

Example 14. The study of the effectiveness of prevention of calves in the proposed method
For the study, experimental and control groups of five animals were formed in each group at the age of seven days. The calves of the experimental group were injected with the proposed method a preparation based on Bac. subtilis at a dose of 50 mg / kg weight after 48 hours for 20 days.

 The animals of the control group were not injected.

 The criteria for evaluating the effectiveness of prevention by the proposed method in comparison with the control were: changes in the level of hemoglobin in the blood, red blood cells, white blood cells, subpopulations of T-lymphocytes and leukogram data; general condition of calves; indicators for the incidence of calves.

 T-lymphocytes were determined using the method of spontaneous rosette formation, based on the interaction of the membrane E-receptor of T-lymphocytes with sheep erythrocytes.

 Research results. In the study of calf blood prior to preventive measures, no characteristic differences in the morphological and immunological parameters of the blood serum of animals of the experimental and control groups were recorded. Analyzes showed low levels of activated T-lymphocytes and post-thymic precursors in the blood of animals.

 Further studies show that in the process of prophylaxis, in both experimental and control calves, quantitative changes in hematological parameters and all populations of T-lymphocytes occur in the direction of their increase, which is due to age-specific characteristics. However, in animals of the experimental group, their changes were more pronounced in comparison with the control.

 In blood test calves 10 days after the start of prophylaxis, compared with the control, the level of leukocytes increases by 6.2%, the content of lymphocytes by 6.6%, T-rock - by 51.85%, p-rock by 43, 3%, BE-rock by 67.2%, BE-rock by 168.2% and aE-rock by 225%.

 On the 20th day of the study, from the studied hematological parameters in comparison with the control, an increase in lymphocytes by 14.6% and leukocytes by 6.3% was recorded in the experimental group, and a blood test for the content of subpopulations of T-lymphocytes showed an increase in the content of te-rock by 18, respectively. 1%, RE-rock by 10.6%, BE-rock by 33.7%, BE-rock by 34.1%, and BE-rock by 40.5%.

 Industrial applicability. The invention can be used in medicine, veterinary medicine and biotechnology.

Sources of scientific, technical and patent information:
1. Auth. testimonial. USSR N 681923, MKI C 12 N 15/00, publ. 1979

 2. Auth. testimonial. USSR N 1722502, MKI A 61 K 39/02, 1989

 3. RF patent N 1839459, MKI C 12 N 1/21, publ. 1995 year

 4. Application EPO N 0287699, MKI C 12 N 1/20, publ. 10.26.88 g.

 5. Auth. testimonial. USSR N 1398868, MKI A 61 K 35/74, publ. 1988 year

 6. Chang S., Cohen S.N. High frequency transformationof Bac. subtilis protoplasts by plasmid DNA.-Mol. Gen. Genet., 1976, v. 168, p. 111 - 115.

 7. Labinskaya A.S. Microbiology with the technique of microbiological research. - M., "Medicine", 1978. - p. 47.

 8. Birnboim H. C., Doly J. A rapid alkaline eatraction procedure for screening recombinant plasmid DNA. - Nucleic Acids Res., 1979. - v. 7, p. 1513 - 1523.

Claims (4)

 1. The bacterial strain Bacillus subtilis VKPM B-7092, used as a component of the drug against viral and bacterial infections.  2. The bacterial strain Bacillus subtilis VKPM B-7048, used as a component of the drug against viral and bacterial infections.  3. The bacterial strain Bacillus licheniformis VKPM B-7038, used as a component of the drug against viral and bacterial infections. 4. The drug against viral and bacterial infections, including a mixture of the biomass of the bacterial strains Bacillus subtilis and Bacillus licheniformis in equal proportions, characterized in that it additionally contains starch and sugar, and as the biomass of the bacterial strains Bacillus subtilis and Bacillus licheniformis use the biomass of bacterial strains Bacillus licheniformis subtilis VKPM B-7092, VKPM B-7048 and Bacillus licheniformis VKPM B-7038 in spore form with a titer of at least 3 • 10 ¹⁰ spores / g in the following quantitative ratio of components, wt.%:
A mixture of spores of bacteria Bacillus subtilis and Bacillus licheniformis in equal proportions - 0.1 - 1.0
Starch - 1.0 - 3.0
Sugar - Else

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