The bacterial strain bacillus subtilis with a broad spectrum of antagonistic activity and antibiotic resistance

 

(57) Abstract:

The invention relates to Microbiology and can be used to obtain preparations of probiotics for the treatment of inflammatory disease and dyspepsia. The strain of Bacillus subtilis 12V isolated from tocompete, characterized by a high antagonistic activity against veterinary strains of pathogenic and conditionally pathogenic microorganisms and antibiotic resistance. Deposited in the collection of pure cultures of microorganisms VGNKI. 6 table.

The invention relates to agricultural Microbiology and represents a new strain of bacteria of the genus Bacillus, which is an antagonist to the veterinary strains of pathogenic microorganisms of the genera: Streptococcus, Esherichia coil, Pseudomonas, Sthaphilococcus, Proteus, Shigella, Hexaera and broad resistance to antibiotics.

Currently, the drugs of probiotics bacteria of the genus Bacillus are widely used in veterinary medicine for treatment of various diseases of poultry and farm animals, because they suppress the development of pathogenic bacteria: AREA (lactoalbumin), cobalin, sporulant [1-3]. Preparations of probiotics on the basis of bacteria of the genus Bacillus have a broad antagonism in is [4].

The main drawback of these drugs is low biological effectiveness against strains of Pseudomonas, Streptococcus, inability concomitant use with antibiotics for severe infection. For the treatment of humans and animals: already created products from bacilli with antibioticassociated that allows them to use on the background of antibiotic therapy to prevent the development of dysbiosis [5, 6].

Closest to the invention is a recombinant strain of B. subtilis VKPM-4759, with a broad spectrum of antagonistic activity against pathogenic microorganisms, but low activity against Pseudomonas aeruginosa (4-6 mm stunted growth of the test strains) and not to delay the growth of Escherihia coli and Streptococcus, which are often the cause of diseases of farm animals [2].

The aim of the invention is a new strain of bacteria of the genus Bacillus, with a broader spectrum of antagonistic activity, including strains of the genera Pseudomonas, Streptococcus, Escherihia coli and antibiotic resistant. This allows you to construct on the basis of the probiotic drug for the treatment of severe forms of infection while antibiotics.

The strain selected organisms VGNKI. Identification of the strain was carried out according to the "Determinant bacteria Bergey M. Mir, 1997.

B. subtilis is a gram-positive aerobic rods that produce catalase, bad spirulina. On wort-agar, agar medium Gause N 2 form a convex, matte, round, small colony, 0.2-0.7 mm in diameter; clear wavy edge, closely adjacent to the nutrient medium. On MPA growth is bad.

In smears from colonies grown on Gause N 2 in 24 hours detected straight rod-shaped cells are small and thin, single or in chains, meet single controversy after 48 hours of growth. On BCH culture forms a homogeneous turbidity and benthic sediment, the film does not form.

Does not grow in anaerobic conditions, hydrolyzes urea and starch, thins gelatine. Gives a positive reaction Voges-Proskauer. Ferments glucose, sucrose, maltose, mannitol, lactose. Reduces nitrates. Produces catalase, protease, lipase. Has no lecithinase activity. Forms ammonia and hydrogen sulfide, does not produce indole. Has mobility.

Culture of B. subtilis 12 B has been verified emissions (virulence, toxicity and toxigenicity).

To study the virulence piece is the principal solution of sodium chloride and prepared bacterial suspension with a cell concentration of from 0.5 to 5.0 billion in ml. The resulting suspension was injected white outbred mice weighing (161) g in two ways: once intraperitoneally and orally for 5 days. The results of the tests are presented in table 1.

For a more complete study of the safety of strain when administered orally, were tested high doses of strain at a single oral administration to mice (table 2). It is established that single injection of peros high doses of strain (10-20)109cells/ml leads to a reduction in the weight of animals on the first day after injection, but in the following days, the weight of the animals recovered.

For toxicity studies (table. 3) strain was grown on mesopatamia agar for 24 hours at 37oC, washed with 0.9% sodium chloride solution, preparing a suspension containing 1.0 to 5.0 billion cells in 1 ml, which then iactiveaware heating at 100oC for 1 hour. The suspension was imposed by the methods described in the study of virulence.

To study oxygenate (PL. 4) strain were grown for 10 days on mesopatamia broth at a temperature of 37oC. Upon expiration of the culture fluid was centrifuged at 8000 rpm for 30 minutes To introduce the mice used nodosa is lugenia for animals held for 5 days after the course of injections or oral administration. During this period, all animals remained active, well ate food rations, had gained weight, physiological functions and behavioral responses have not changed. The obtained data testify to the safety of the strain.

To determine the specific activity investigated the antagonistic activity of strain in relation to the test cultures. The study was carried out by the method trimmed antagonism. Culture strains were sown loop in the centre of Petri dishes with agar medium Gause N 2. Crops were incubated in a thermostat at 37oC for 72 hours. Then grown culture was podseval stroke test microorganisms (500 millionth suspension subsistence crops in saline solution). Analysis was performed after 18 h incubation at 37oC largest zones of no growth of the test cultures. Control of the growth of the test cultures was parallel growing them on plates with agar medium Gause N2 without culture of the studied strain. The data obtained test results are presented in table 5.

The culture of strain is characterized by a high antagonistic activity against wide spectrum of pathogenic and conditionally pathogenic microorganismal standard disks, impregnated with antibiotics. The strains were sown on agar nutrient medium and played discs with antibiotics, 5 per Petri dish. Crops were incubated at 37oC for 24 hours of the sensitivity of the cultures to antibiotics was expressed in mm diameter of zone of growth inhibition. Resistant believed strains, zones of growth inhibition which was - (0-5) mm; insensitive (6-10 mm) sensitive (11-20) mm; high-sensitivity - more than 22 mm [7]. The results of the study are presented in table 6.

From the table it follows that the strain B. subtilis 12B has a broad resistance to antibiotics, including lincomycin, tetracycline, monomitsina, polymyxin, oxacillin, streptomycin, ampicillin, benzylpenicillin.

Thus, isolated a new strain of B. subtilis 12V, which is an antagonist to the veterinary strains of pathogenic microorganisms of the genera: Streptococcus, Esherichia coli, Pseudomonas, Sthaphilococcus, Proteus, Shigella, Hexaera with a broad resistance to antibiotics, that creates the possibility of designing on the basis of preparations of probiotics for concomitant use with antibiotics.

Sources of information

1. Ivanovo A. A., Veprev N. S., Dimireva Centuries, Lagunova O. P. With Whom.In., Reznik, S. P., Sorokulova I. B. and other Preventive biological preparation of sublin /RU Patent N 2035185, publ. 31.01.92. Bull. N 14, A 61 K 35/66.

3. Reznik, S. R.. Sorokulova I. B., Kunicka C. A. and other Preventive biological sporulant //Patent N 2035186. EN. A 61 K 35/56, publ. 20.05.95. Bull. N 14.

4. Blinkova L. P., Semenova, S. A., Butova L., and other Antagonistic activity of freshly isolated strains of bacteria of the genus Bacillus //IMEI. -1994. -N 5. - S. 71-75.

5. Bulantsev A. L., N. Tikhonov.G., Lipnitsky A. C. Strain of Bacillus subtilis PAC resistant to rifampicin, ampicillin and streptomycin, which have antibacterial activity against pathogenic microorganisms //Pat. RF N 2115725, C 12 N 1/20. - Publ. 20.07.98. Bull. N 20.

6. Zhirkov I. N., Bratukhin And. And. the Application of probiotic RACES for the correction of dysbacteriosis in calves //veterinary medicine. -1999. - #4. -S. 40-42.

7. Birger M. O. Handbook of microbiological and virological methods of examination. Determination of the sensitivity of microorganisms to antibiotics. -M.: Medicine, 1982. -S. 180.

The bacterial strain Bacillus subtilis 12V-DEPT collection of microorganisms VGNKI with a broad spectrum of antagonistic activity and antibiotic resistance.

 

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