RU2444366C1 - Antibiotic resistant therapeutic biological product for bacterial and mycotic infections - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science and biotechnology. A biological product contains mixed biomass of two Bacillus subtilis strains, a stabiliser or an excipient, or a carrier. The biological product is presented in dry or liquid form and contains mixed biomass of the B.subtilis 11B strain and the Bacillus subtilis 12B strain in equal proportions with titre not less than 1×10⁸ CFU/g or 1×10⁸ CFU/ml respectively in the amount of 25-50 or 91 wt %, the stabiliser or the excipient, or the carrier - the rest.

EFFECT: biological product possess a wide antagonistic spectrum to various pathogenic bacteria and fungi, as well as resistance to a wide range of antibiotics.

3 cl, 4 tbl, 3 ex

Description

The invention relates to microbiology, veterinary medicine and biotechnology and is a new drug - a probiotic from two strains of bacteria of the genus Bacillus, which has a wide spectrum of antagonism against various pathogenic types of bacteria and fungi, as well as resistance to a wide range of antibiotics.

Currently, preparations - probiotics from bacteria of the genus Bacillus - are widely used in veterinary medicine for the treatment of various diseases of birds and farm animals, since they inhibit the development of pathogenic bacteria. However, in severe infectious diseases, it is sometimes impossible to refuse to prescribe antibiotics, since they have the fastest therapeutic effect. The use of antibiotics leads to negative consequences for the animal organism: the development of dysbiosis and a decrease in immunity, as well as the development of antibiotic resistance in pathogenic strains. To remove the negative effects of antibiotic therapy, probiotics are additionally used, the earlier the probiotic is used, the less complications after antibiotic therapy are. It is optimal to use a probiotic simultaneously with an antibiotic, but for this, the strains that make up the probiotic preparation must be resistant to antibiotics.

A natural strain of Bacillus subtilis 11B is known, which has a wide spectrum of antagonism against bacterial and fungal pathogens that cause diseases of humans, animals and plants, which makes it possible to create probiotic preparations based on it that can treat a wide range of diseases and can be used in medical, veterinary and agricultural practice (one).

The drug based on the Bacillus subtilis 11B (2) strain is resistant to penicillin antibiotics (b-lactam antibiotics: penicillin, carbenicillin, ampicillin), polymyxin, and is also insensitive to streptomycin, gentamicin, tetracycline. Therefore, it is possible to prescribe a drug for the treatment of people and animals together with antibiotics, to which the strain of Bacillus subtilis 11B is resistant, to prevent complications of antibiotic therapy (the development of intestinal dysbiosis and decreased immunity).

Natural Bacillus subtilis 12B (3) and a preparation based on it (4), used as a veterinary probiotic, are also known. The strain and the drug are antagonistic to pathogenic veterinary strains of pathogenic bacteria and are resistant (or low sensitivity) to a number of antibiotics, including tobramycin, erythromycin, lincomycin, neomycin, rifampicin, i.e. to those antibiotics to which the Bacillus subtilis strain is sensitive 11B.

The combination of two strains of Bacillus subtilis 11B and Bacillus subtilis 12 in one drug allows you to expand the spectrum of antibiotic resistance of the drug and prescribe the drug together with antibiotics, without compromising its effectiveness.

To increase the effectiveness of the action of drugs - probiotics from bacilli - complex preparations are created, including several strains of bacteria. Closest to the invention are strains of Bacillus subtilis and Bacillus liheniformis, used as components of the drug against viral and bacterial infections, and a drug based on these strains (5).

The bacterial strains of Bacillus Sabtilis VKPM B-7092, VKPM B-7048 and the bacterial strain Bacillus licheniformis VKPM B-7038 are used as components of Vetom against viral and bacterial infections. The preparation contains a mixture of biomass of these strains in spore form with a titer of at least 3 × 10 ¹⁰ spores / g. The composition of the drug also includes starch and sugar with a certain quantitative ratio of components. The complex bacterial preparation has a wide spectrum of antiviral and antibacterial activity.

The disadvantage of this drug is the lack of antagonism in this drug against mold fungi of the genera Fusarium, Alternaria, Penicillium - sources of dangerous mycotoxins, spores of which enter animal feed and can cause mycotoxicosis. There is no indication of the possibility of using the drug in conjunction with antibiotics, without which it is impossible to do with severe forms of infection.

The objective of the invention is the creation of a new complex bacterial preparation - a probiotic based on the bacterial strains Bacillus subtilis 11B and Bacillus subtilis 12B, which has a wider spectrum of antagonistic activity, including strains of the genera Pseudomonas, Streptococcus, Escherihia coli, fungi of the genera Fusarium, Alternaria , and resistant to a wide range of antibiotics, which allows it to be used to treat severe infections with simultaneous therapy with an antibiotic.

To solve this problem are offered:

- the bacterial strain Bacillus subtilis 11B, deposited under the number VKM B-2218D in the All-Russian collection of microorganisms of the IBPM RAS on 08.19.1999. The strain was isolated from a healthy wheat plant in the Ufa region. Identification of the strain was carried out according to the “Determinator of bacteria Bergie”. - M .: Mir, 1997;

- B. subtilis strain 12B, deposited in the collection of microorganisms VGNKI under the number 12B-DEPT. The strain isolated from zoological compost in the Ufa region of the Republic of Bashkortostan. Identification of the strain was carried out according to the “Determinator of bacteria Bergie”. - M .: Mir, 1997.

The technical result is that the preparation contains biomass of strains of bacteria Bacillus subtilis 11B and bacteria Bacillus subtilis 12B in equal proportions.

The dry form of the drug additionally contains starch and sugar.

The preparation contains living cells of the strain Bacillus subtilis 12B and the strain Bacillus subtilis 11B in equal amounts and a stabilizer (or carrier) in the following ratio of components, wt%:

The biomass of strains of Bacillus subtilis, 1 × 10 ⁸ -10 × 10 ⁹ live microbial cells in 1 ml of the drug 90-99 Stabilizer, filler (carrier) 1-10

As a stabilizer for the liquid form of the drug, sodium chloride at a concentration of 0.9% can be used. Upon receipt of dry forms of the drug (granulation, tabletting or freeze drying), a filler (or carrier) is introduced into the biomass, due to its protective effect for bacterial cells upon drying.

Upon receipt of dry biomass of strains (to obtain powder or granules), gluconate or calcium stearate, starch, gelatin and sucrose and other stabilizers are additionally added to the biomass in an amount of 1.0-10.0 wt.% In order to give the biomass the necessary physical properties . If necessary, you can mix the finished biomass with a sorbent (for example, activated carbon, carbolong, lactulose, acetylphthalyl cellulose, aluminum phosphate gel) in the ratio of 1: 1 by dry residue, after which the liquid is separated by filtration and the microgranules of the immobilized biomass are dried by vacuum drying to a residual moisture content of 5- 10%.

After obtaining the biomass in dry form (powder, granules), the biomass is mixed with auxiliary substances in a ratio of 2: 1 - 3: 1 and tableted. As auxiliary substances use (wt.%.): Calcium stearate (0.5-1.0), talc (2.5-3.0) and (or) starch (0.5-1.0) and ( or) aerosil (0.5-0.8), glucose (5.0-20.0), milk powder (10.0-30.0) and (or) methyl cellulose (or polyvinylpyrrolidone) (0.1-0 ,5). Get a tablet form of the drug, convenient to use.

Example 1. On the basis of strain B.subtilis 11B with typical morphological, cultural, biochemical properties, 5 variants of a liquid preparation with different contents of microbial cells in the composition of 1 dose of the preparation were prepared.

Option 1. A preparation containing, wt.%:

B.subtilis 11B biomass, 0.5 × 10 ⁸ live microbial cells or colony forming units (CFU);

B.subtilis 12B biomass, 0.5 × 10 ⁸ live microbial cells or colony forming units (CFU) in 1 ml of the drug 91; stabilizer (sodium chloride) 9.

Option 2

A preparation containing, wt.%:

B.subtilis 11B biomass, 2 × 10 ⁸ live microbial cells (CFU);

B.subtilis biomass 12B, 2 × 10 ⁸ live microbial cells (CFU) in 1 ml of the drug 91, stabilizer (sodium chloride) 9.

Option 3. A preparation containing, wt.%:

B.subtilis 11B biomass, 5 × 10 ⁸ live microbial cells (CFU);

B.subtilis 12V biomass, 5 × 10 ⁸ live microbial cells (CFU) in 1 ml of the drug 91; stabilizer (sodium chloride) 9.

Option 4

A preparation containing, wt.%:

B.subtilis 11B biomass, 1.0 × 10 ⁹ live microbial cells (CFU);

B.subtilis 12B biomass, 1.0 × 10 ⁹ live microbial cells (CFU) in 1 ml of the drug 91; stabilizer (sodium chloride) 9.

Manufactured variants of the drug were poured into sterile vials, corked with rubber stoppers and dipped in aluminum caps.

Example 2. The strain B. subtilis 11B is cultivated in a liquid nutrient medium that provides spore formation for 16 hours.

Strain B. subtilis 12B is cultivated in a liquid nutrient medium that provides spore formation for 16 hours.

The resulting biomass is mixed in equal proportions (if necessary, concentrated by microfiltration to a microbial cell content of 100 × 10 ⁹ cells / ml by TOC for 10 units). A stabilizer based on starch and lactose or a sucrose-gelatin medium is added to the biomass, mixed, granulated and dried to a residual moisture content of not more than 5%. Next, the powder is ground to a homogeneous mass and dosed into bottles (or bags).

Option 5. A preparation containing, wt.% .:

biomass 25.0 starch 31.1 lactose 42.6 methyl cellulose (or polyvinylpyrrolididone) 1.3

The finished powder is white or beige, sweetish in taste. Residual humidity is not more than 5%. The powder is soluble in water, 0.1 N. HCl and neutral solutions for 15 min with the formation of a loose lumpy precipitate. The finished powder contains B. subtilis biomass in an amount of at least (1.5 ± 0.5) × 10 ⁸ CFU / g.

Example 3. The strain B. subtilis 11B is cultured in a liquid nutrient medium that provides spore formation for 16 hours.

Strain B. subtilis 12B is cultivated in a liquid nutrient medium that provides spore formation for 16 hours.

The resulting biomass is mixed in equal proportions (if necessary, concentrated by microfiltration to a microbial cell content of 100 × 10 ⁹ cells / ml by TOC for 10 units). A stabilizer based on starch and lactose or a sucrose-gelatin medium is added to the biomass and calcium gluconate is added in an amount of 2.0 wt.%, Mixed and dried in an open way (or freeze-dried in cassettes). At the end of drying, the powder is ground to a homogeneous mass, auxiliary substances are added and tableted.

Option 6. A preparation containing, wt.%:

biomass 50,0 calcium stearate 0.8 sucrose-gelatin medium 29.6 calcium gluconate 2.0 talc 2.5 glucose 5,0 powdered milk 10.0 cellulose 0.1

The finished tablets have a round shape with a diameter of 0.8-12 mm with a smooth hard surface of whitish-gray or yellowish color, sweetish in taste, with a specific smell. The average tablet weight is from 0.75 ± 0.05 g with a residual moisture content of not more than 5%. The tablets dissolve in 0.1 n. HCl and neutral solutions for 30-45 min with the formation of a loose lumpy precipitate. The finished tablets contain the biomass of the strain B. subtilis in the amount of (2.5 ± 0.5) × 10 ⁸ KOE.

The safety of the obtained variants of the drug in laboratory animals, specific antagonistic activity against test cultures — representatives of various groups of pathogenic and conditionally pathogenic microorganisms, and antibiotic resistance were tested.

The drug is characterized by harmlessness (see table 1).

To determine the safety, the contents of the bottle with the liquid preparation were used without dilution, the powder or tablet was diluted in 10 ml of physiological saline and 0.5 ml of the drug was administered orally to mice. For each experiment, at least 10 mice weighing 15–16 g were used. The drug was considered harmless if all the mice remained alive for five days of observation and no disease was detected.

Table 1 The study of the safety of the drug No. Variants of the drug (number of cells in 1 dose) The number of animals (pieces) The average weight of animals, g It was Survived Before introduction After 1 day After 5 days one 0.5 × 10 ⁸ 10 10 15.4 ± 0.3 15.6 ± 0.3 16.9 ± 0.3 2 2.0 × 10 ⁸ 10 10 15.7 ± 0.2 15.9 ± 0.3 17.0 ± 0.2 3 5.0 × 10 ⁸ 10 10 15.82 ± 0.2 16.0 ± 0.2 17.08 ± 0.2 four 1.0 × 10 ⁹ 10 10 15.65 ± 0.2 15.7 ± 0.2 17.1 ± 0.2 5 1.5 × 10 ⁸ 10 10 16.0 ± 0.2 16.1 ± 0.2 17.2 ± 0.2 6 2.5 × 10 ⁸ 10 10 15.6 ± 0.2 15.8 ± 0.2 17.1 ± 0.2

The test showed that all animals remained alive, their initial mass increased, there were no signs of the disease.

The drug is characterized by a wide spectrum of antagonistic activity against test strains of cultures of pathogenic microorganisms (table 2).

To determine the specific activity, the antagonistic activity of the drug variants with respect to test cultures was investigated. The study was carried out by the method of delayed antagonism. To do this, the contents of the powder bottle (or tablet) was dissolved in 2-5 ml of physiological saline. The resulting suspension was seeded by a stroke along the diameter of a Petri dish with Gauze N 2 agar medium. Crops were incubated in a thermostat at 37 ° C for 72 hours. Then, microorganisms were inoculated with a stroke (500-ml suspension of daily cultures in physiological saline). The results were taken into account after 18 hours of incubation at 37 ° C according to the size of the zones of lack of growth of test cultures.

The growth control of test cultures was served by their parallel growth on plates with agarized Gauze medium No. 2 without the studied culture.

table 2 Antagonistic activity of drug variants No. Test culture Variants of the drug (number of cells in 1 dose) one 2 3 four 5 6 0.5 × 10 ⁸ 2.0 × 10 ⁹ 5.0 × 10 ⁹ 1.0 × 10 ⁹ 1.5 × 10 ⁹ 2.5 × 10 ⁸ one 2 3 four 5 6 7 8 2 Staphylococcus 10-15 15-20 15-20 15-20 18-20 18-20 aureus (Filippov) 3 Staphylococcus 10-15 15-20 15-20 15-20 18-20 18-20 aureus (Nikiforov) four St. aureus 66 10-12 15-16 15-16 15-16 15-16 15-16 5 St. aureus 67 8-10 10-12 10-12 10-12 10-12 10-12 7 St. aureus 80 10-12 12-15 12-15 12-15 12-15 12-15 8 Proteus vulgaris 177 10-15 15-20 15-20 15-20 15-20 15-20 9 Pr. vulgaris 12 8-10 10-12 12-15 12-15 12-15 12-15 10 P. vulgaris 48 8-10 12-15 12-15 12-15 12-15 12-15 eleven Pr. vulgaris 41 8-10 10-12 12-15 12-15 12-15 12-15 12 Proteus mirabilis 237 15-18 18-20 18-20 18-20 18-20 18-20 13 Pr. mirabilis 40 12-15 15-20 15-20 15-20 15-20 15-20 fourteen Pr. mirabilis 36 12-15 15-18 15-18 15-18 15-18 15-18 fifteen Pr. mirabilis 35 15-18 18-20 18-20 18-20 18-20 18-20 16 Candida albicans 25-30 25-30 25-30 25-30 25-30 25-30 17 Escherihia coli 177 20-22 22-25 22-25 22-25 22-25 22-25 eighteen E.coli 154 10-12 12-15 12-15 12-15 12-15 12-15 19 E.coli 140 10-12 12-15 12-15 12-15 12-15 12-15 twenty Shigella sonne 2586 10-12 12-15 12-15 12-15 12-15 12-15 21 Shigella hk 20-22 22-25 22-25 22-25 22-25 22-25 22 Pseudomonas 12-15 15-20 15-20 15-20 15-20 15-20 aeruginosa 8 23 P. aeruginosa 126 10-12 12-15 12-15 12-15 12-15 12-15 24 Streptococcus 321 10-12 12-15 12-15 12-15 12-15 12-15 25 Streptococcus 325 10-12 12-15 12-15 12-15 12-15 12-15 26 Streptococcus 191 10-12 12-15 12-15 12-15 12-15 12-15 27 Paratyhv 269 10-12 12-15 12-15 12-15 12-15 12-15 28 Paratyhv 15 12-15 15-20 15-20 15-20 15-20 15-20 29th Hexaere II 1230 12-15 15-20 15-20 15-20 15-20 15-20

From the data of table 1 it follows that the optimal number of living microbial cells in a single dose of the drug is 2-5 × 10 ⁸ . A decrease in the number of microbial cells to 0.5 × 10 ⁸ reduces the level of antagonistic activity, and an increase in the number of microbial cells to 10 × 10 ⁸ and higher does not significantly change the antagonistic activity of the drug in relation to test cultures of microorganisms. The use of the drug in tablets (option 6) does not reduce the level of antagonistic activity.

The drug is resistant to a number of antibiotics (table 3).

The sensitivity of the drug to antibiotics was determined using standard disks impregnated with antibiotics. In a Petri dish with MPA, 1 ml of a cell suspension of the drug in a 0.9% sodium chloride solution was poured and evenly distributed over the surface of the agar. Excess was removed with a Pasteur pipette. Disks with antibiotics were laid out on the surface of seeded agar with tweezers, 5 disks per cup. The cups were kept in a thermostat at a temperature of (37 ± 1) ° С for (18 ± 2) h, after which growth inhibition zones around the disks were measured, including the diameter of the disk itself.

Table 3 Antibiotic resistance of the drug options No. Antibiotics Growth inhibition zones, mm Drug options one 2 3 four 5 6 one Ampicillin 0-6 0-6 0-6 0-6 0-6 0-6 2 Benzylpenicillin 0 0 6 8 6 6-8 3 Carbenicillin 0 0 0 0 0 0 four Polymyxin 0 0 0 0 0 0 5 Streptomycin 0-8 0-8 0-8 0-8 0-8 0-8 6 Rifampicin 0-6 0-6 6 6 6-8 6-8 7 Gentamicin 0-7 0-7 0-6 0-6 0-6 0-6 8 Clotrimazole 8-10 8-10 8-10 10 10 10 9 Levomycetin 18-20 18-20 18-20 18-20 18-20 18-20 10 Tetracycline 6-8 6-8 6-8 6-8 6-8 6-8 eleven Monomycin 6-8 6 6 6-8 6-8 8 12 Heptomycin 10-15 10-15 10-15 10-15 10-15 10-15 13 Clotrimazole 10-12 10-12 10-12 10-12 10-12 10-12 fourteen Neomycin 6 6 0 6-8 6-8 6-8 fifteen Teparin 15-20 15-20 15-20 15-20 15-20 15-20 16 Kanamycin 10-15 10-15 10-15 10-15 10-15 10-15 17 Novogram 20-25 20-25 20-25 20-25 20-25 20-25 eighteen Bisengiol 10-15 10-15 10-15 10-15 10-15 10-15 19 Tobramycin 6-8 0-8 0 6 6-9 6 twenty Erythromycin 6-8 0 6 6 6-9 6-8

From the data obtained, it follows that the drug is resistant to the action of penicillin antibiotics (b-lactam antibiotics: penicillin, carbenicillin, ampicillin), polymyxin, and is also insensitive to streptomycin, gentamicin, tetracycline, tobramycin, erythromycin, monomycin, romycin.

The effectiveness of the claimed drug in optimal concentrations of microbial cells (2-10) × 10 ⁸ for the normalization of intestinal microbiocenosis was evaluated in the treatment of 10 sick calves with severe dyspepsia. The drug was prescribed 1 dose twice a day for 10 days. The results of the study of intestinal microflora in sick calves before treatment and 7-10 days after treatment are presented in table 4.

After prescribing the drug, the symptoms of dyspepsia and diarrhea were stopped within 3-7 days, the drug was given for 10 days.

Indicators of the state of intestinal microbiocenosis show that the therapeutic effect of the drug is the effective elimination of UPM (98.0%) and the restoration of normal flora (90%). Its absolute harmlessness and good tolerance are shown.

Table 4 The state of intestinal microflora in sick calves during treatment with a complex drug Representatives of the intestinal microflora Number of patients
(n-30) Before treatment After treatment Abs. % Abs. % Decrease in bifidobacteria (10 ⁴ -10 ⁵ ) thirty one hundred four 13.3 Lactobacillus reduction (10 ⁴ -10 ⁵ ) 22 73.3 2 6.6 Decreased spore anaerobes (10 ³ -10 ⁴ ) eighteen 60 four 13.3 UPF increase (10 ⁶ ), including: Proteus fourteen 46.7 0 0

Klibsiella 9 30,0 0 0 Hemolytic Escherichia fourteen 46.7 0 0 Staphylococci eighteen 60 3 1,0 Pseudomonads 6 20,0 0 0 Streptococcus 12 40,0 0 0 Lactose-negative enterobacteria 9 30,0 3 1,0 Candida Fungi 6 20,0 0 0 Salmonella four 13.3 0 0 UPM elimination efficiency 98.0% Normoflora recovery efficiency 90%

Information sources

1. Patent No. 2182172 "The strain of bacteria Bacillus subtilis with a wide spectrum of antagonistic activity", publ. 10/13/2002, bull. No. 13.

2. Patent No. 2181596 "A drug from bacteria of the genus Bacillus", publ. 04/27/2002, bull. No. 12.

3. Patent No. 2169767 "Bacillus subtilis bacterial strain having a wide spectrum of antagonistic activity and antibiotic resistance", publ. 06/27/2001, bull. Number 18.

4. Patent No. 2172175 "Medical and preventive biological product", publ. 08/20/2001, bull. Number 23.

5. Patent No. 2142287 "Bacterial strains of Bacillus subtilis and Bacillus licheniformis used as components of the drug against viral and bacterial infections, and a drug based on these strains", publ. 12/10/1999.

Claims (3)

1. The treatment and prophylactic biological product against bacterial and fungal infections, having antibiotic resistance, comprising a mixture of biomass of two strains of bacteria Bacillus subtilis, a stabilizer, or a filler, or a carrier, characterized in that it is made in dry or liquid form and contains a mixture of biomass of strain B. subtilis 11B and the biomass of the strain Bacillus subtilis 12B in equal proportions with a titer of at least 1 × 10 ⁸ CFU / g or 1 × 10 ⁸ CFU / ml in an amount of 25-50 or 91 wt.%, stabilizer, or filler, or carrier rest. 2. The treatment and prophylactic biological product according to claim 1, characterized in that it is dry, contains a mixture of biomass of strain B. subtilis 11B and Bacillus subtilis 12B in equal proportions with a titer of at least 1 × 10 ⁸ CFU / g and a stabilizer? or filler or carrier in the following ratio of components, wt.%:
A mixture of biomass bacteria strain B. subtilis 11B and Bacillus subtilis 12B in equal proportions with a titer of at least 1 × 10 ⁸ CFU / g 25-50 Stabilizer, or filler, or carrier rest 3. The treatment and prophylactic biological product according to claim 1, characterized in that it is made in liquid form, contains a mixture of biomass of bacteria of the strain B. subtilis 11B and Bacillus subtilis 12B in equal proportions with a titer of at least 1 × 10 ⁸ CFU / ml, and as a stabilizer contains sodium chloride in the following ratio, wt.%:
A mixture of biomass bacteria strain B.subtilis 11B and Bacillus subtilis 12B in equal proportions with a titer of at least 1 × 10 ⁸ CFU 91 Sodium chloride 9