RU2181596C1 - Curative preparation of bacillusstrain bacteria - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: the biopreparation contains the strain of Bacillus subtilis 11V VKM V-2218D, a solvent and a filler. EFFECT: increased antagonistic activity regarding a wide range of pathogenic and conditionally pathogenic microorganisms, improved resistance to a variety of antibiotics. 4 tbl, 1 ex

Description

 The invention relates to medicine and relates to the creation of new drugs from bacteria of the genus Bacillus.

 Traditional therapy for bacterial diseases involves the widespread use of antibiotic drugs. However, these drugs are often toxic, have a nonspecific bactericidal effect, including suppress the endogenous microflora, which leads to the development of various complications, for example, dysbiosis. Therefore, recently proposed new antibacterial drugs that have a pronounced activity against pathogenic and conditionally pathogenic bacteria and do not violate human microbiocenoses. Preparations based on bacteria of the genus Bacillus have a good therapeutic effect for the treatment of gastrointestinal, purulent-inflammatory, and urogenital diseases, which have comparable antibiotic efficacy and do not give complications in the form of allergies and dysbacterioses (1-3).

 The composition of probiotic preparations based on bacteria of the genus Bacillus includes strains with a different spectrum of antibacterial and antifungal activity. The effectiveness of the drugs is determined by the breadth of the spectrum and the strength of the antibacterial activity of the strain included in the drug.

 The closest analogue of the invention is the therapeutic and prophylactic biological product Baktisporin based on the antagonistically active strain B. subtilis 3H for the treatment of a wide range of diseases, produced in ampoules and used orally in the form of a solution (4).

 The disadvantage of bactisporin is the lack of biological effectiveness against strains of the genera Pseudomonas, Streptococcus, Paratyhv, which are often the cause of the development of acute intestinal infections, dysbiosis or aggravate their course.

 The problem solved by the invention is the creation of a new probiotic preparation with a wide spectrum of antagonistic activity, including against strains of the genera Pseudomonas, Streptococcus, Paratyhv, the use of which is possible with antibiotics in order to prevent the development of dysbiosis.

 This is achieved by the fact that the Bacillus subtilis 11B strain is used as the basis of the biological product. The strain was deposited under the number VKM V-2218D in the All-Russian collection of microorganisms of the IBPM RAS 08/19/1999.

 The strain culture is characterized by the following properties: B.subtilis VKM B-2218 D - gram-positive aerobic spore-forming bacilli producing catalase. On meat-peptone agar (MPA), wort agar give abundant growth. On MPA - it forms grayish-white small-wrinkled colonies with wavy edges, slightly growing into agar, of a viscous consistency. Growth in a liquid medium is accompanied by turbidity of the medium, the formation of a film and sediment. After 18 hours of growth, straight rod-shaped cells with a size of 2-3 • 0.6 μm, located singly, in pairs or in a chain, are found in the smears of the culture. The cells are motile. When spore formation, the cell does not swell. The spores are oval, size 0.9 • 0.6 microns, located in the cell eccentrically.

 The culture ferments glucose, β-galactose, lactose, sucrose and maltose. Does not decompose mannitol, inositol, sorbitol and arabinose. It gives a positive Voges-Proskauer reaction, hydrolyzes starch, gelatin, does not hydrolyze urea, utilizes sodium citrate with glucose, does not use propionate and sodium malonate, and grows weakly under anaerobic conditions. It is characterized by the presence of arginine dehydrolase, lysine decarboxylase, the absence of phenylalanyl deamylase, ornithine decarboxylase. Forms acetylmethylcarbinol. Reduces nitrates, does not form gas on a medium with nitrates under anaerobic conditions. Does not form indole and hydrogen sulfide.

 The strain Bacillus subtilis 11B is characterized by high antagonistic activity against a wide range of pathogenic and conditionally pathogenic microorganisms and resistance to a number of antibiotics.

The preparation contains living cells of Bacillus subtilis 11B and an excipient in the following ratio of components, wt. %:
Biomass of Bacillus subtilis 11B - 1 • 10 ⁹ -5 • 10 ⁹ live microbial cells in 1 ml of solvent - 92-95
Filler - 5-8
As a solvent, physiological saline, distilled or boiled water can be used. As a filler, the preparation may contain 5% lactose or 8% sucrose-gelatin mixture (7% sucrose and 1% gelatin). The need for a filler is due to its protective effect for bacterial cells during further freeze drying of the drug.

 Example. Based on the B.subtilis 11B strain with typical morphological, cultural, biochemical properties, 5 variants of the preparation with different contents of microbial cells in the preparation were prepared.

Option 1. A preparation containing, wt.%:
Biomass of B. subtilis 11B - 1 • 10 ⁸ live microbial cells in 1 ml of physiological saline - 92
Filler - sucrose-gelatin mixture - 8
Option 2

A preparation containing, wt.%:
B. subtilis 11B biomass - 1 • 10 ⁹ live microbial cells in 1 ml of physiological saline - 93
Filler - sucrose-gelatin mixture - 7
Option 3. A preparation containing, wt.%:
B. subtilis 11B biomass - 5 • 10 ⁹ live microbial cells in 1 ml of physiological saline - 94
Filler - sucrose-gelatin mixture - 6
Option 4. A preparation containing, wt.%:
B.subtilis 11B biomass - 10 • 10 ⁹ live microbial cells in 1 ml of physiological saline - 95
Filler - sucrose-gelatin mixture - 5
Option 5. A preparation containing, wt.%:
B.subtilis 11B biomass - 5 • 10 ¹⁰ live microbial cells in 1 ml of physiological saline - 95
Filler - Lactose - 5
Manufactured variants of the drug were poured into sterile vials, dried freeze-dried, corked with rubber stoppers and dipped in aluminum caps. 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 (table 1).

 To determine the safety, the contents of the vial were diluted in 0.5 ml of physiological saline and this dose 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.

 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 range 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. For this, the contents of the vial were dissolved in 1 ml of physiological saline. The resulting suspension was seeded by a stroke along the diameter of the Petri dish with Gauze 2 agar medium. Crops were incubated in a thermostat at 37 ^° C for 72 hours. Then, microorganisms were inoculated with a stroke (500 million suspension of daily cultures in physiological saline). Analysis was carried out after 18 hours of incubation at 37 ^o With the size of the zones of lack of growth of test cultures.

 The growth control of test cultures was controlled by their parallel growth on plates with agarized Gauze 2 medium without the studied culture.

From the data table. 1 it follows that the optimal number of living microbial cells in a single dose of the drug is 1-5 • 10 ⁹ . A further increase in the number of microbial cells does not significantly change the antagonistic activity of the drug against test cultures of microorganisms.

 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 preparation 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) ^o С for (18 ± 2) h, after which growth inhibition zones around the disks were measured, including the diameter of the disk itself.

 From the data obtained it follows that the drug is resistant to penicillin antibiotics (b-lactam antibiotics: penicillin, carbenicillin, ampicillin), as well as streptomycin, polymyxin, gentamicin and rifampicin.

The effectiveness of the proposed drug in optimal concentrations of microbial cells (1-5) • 10 ⁹ for the normalization of intestinal microbiocenosis was evaluated in the treatment of 15 patients with acute intestinal infections (OCI) and chronic diseases of the gastrointestinal tract (prolonged course of acute intestinal infections, salmonellosis, chronic colitis, enterocolitis , fermentopathy, intestinal dysbiosis against the background of chronic gastritis, cholecystopancreatitis, chronic hepatitis) with bacterially confirmed bifidoflora deficiency. The drug was prescribed 1 dose twice a day for 10 days. The results of the study of intestinal microflora in patients before and after 7-10 days after treatment are presented in table. 4.

 From the table. 4 it follows that dysbiosis was due to both a decrease in normoflora (bifidobacteria, lactobacilli) and an increase in conditionally pathogenic microflora (UPM). The therapeutic effect of the drug is the effective elimination of UPM - 97.3% and the restoration of normoflora - 90%. Its absolute safety and good tolerance are shown.

Sources of information
1. Smirnov V.V., Reznik S.R., Sorokulova I.B. and others. The drug biosporin for the prevention and treatment of gastrointestinal diseases of a person // A. S. 1722502. - SU. - A 61 K 39/02, publ. 03/30/92.

 2. Smirnov V.V., Reznik S.R., Sorokulova I.B. and other Preventive biological product subalin // Patent 2035185, RU. - A 61 K 35/66, publ. 05/20/95, bull. 14.

 3. Belyavskaya V. A., Sorokulova I. B., Masycheva V. A. Recombinant probiotics: problems and prospects of use for medicine and veterinary medicine // Dysbacteriosis and eubiotics: Abstracts of the All-Russian scientific and practical conference. - M. - 1996. - S. 7.

 4. Mikhailova N. A., Kuznetsova T. P., Kunyagina O. V. Therapeutic and prophylactic drug Baktisporin // Patent 2130316, A 61 K 35/66, prior. 01/23/97.

Claims (1)

A therapeutic and prophylactic biological product, including the live microbial mass of Bacillus subtilis, a solvent and a filler, characterized in that it contains B. subtilis 11B biomass (VKM B-2218D at the IBPM RAS) in the following ratio of components, wt. %:
Bacillus subtilis 11B biomass of living microbial cells - (1-5) • 10 ⁹ in 1 ml of solvent - 92 - 95
Filler - 5 - 8

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