RU2509149C1 - Bacillus subtilis subsp Subtilis STRAIN HAVING APPARENT ANTAGONISM IN RELATION TO Escherichia coli, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes AND RESISTANCE TO STREPTOMYCIN AND TETRACYCLINE - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: bacillus subtilis subsp.subtilis BKM B-2711D strain has apparent antagonism in relation to Escherichia coli, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes, and resistance to streptomycin and tetracycline antibiotics. It is deposited in the All-Russia Collection of Microorganisms of the Institute of Biochemistry and Physiology of Microorganisms Named after G.K. Skryabin of the Russian Academy of Science (IBFM RAN) and has the following registration number: BKM B-2711D, and can be used at production of probiotic bacterial preparations that can be used in veterinary medicine.

EFFECT: invention allows increasing protease and xylanase activities.

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Description

The invention relates to the field of biotechnology, namely to probiotic bacterial preparations that may find application in veterinary medicine.

Bacterial preparations based on strains of microorganisms with antagonistic activity against enteropathogenic bacteria are probiotic.

In the classical studies of I.A. Mechnikov, these properties were studied using lactic acid bacteria as an example. Then the range of producers of probiotic drugs was expanded.

Probiotics based on strains of spore-forming bacteria have recently begun to be used in veterinary medicine. The antagonistic properties of these bacteria attract the attention of many developers.

The most promising producers are strains that have antagonistic activity not only against enteropathogenic bacteria, but also to a wide range of test organisms that cause extraintestinal infections.

The difficulty in treating infectious diseases lies in the occurrence of antibiotic resistance in pathogens. In addition, antibiotics themselves often have a side effect on the human body and cause intestinal dysbiosis, aggravating the course of the underlying disease. This necessitates the search for new approaches to solving this problem.

Probiotics based on bacteria of the genus Bacillus, which suppress the development of pathogenic microorganisms, are widely used in agriculture for the suppression of phytopathogenic bacteria and fungi, in medicine, veterinary medicine for the treatment of various diseases of birds and farm animals [1].

Currently, probiotic preparations from bacteria of the genus Bacillus are widely used in veterinary medicine for the treatment of various diseases of birds and farm animals. Numerous variants of probiotics from spore-forming bacteria have been proposed: bactisporin [5], bacillosporin [6], sporolact [7], sporobacterin [8], subalin [9].

To increase the effectiveness of the prevention and treatment of dysbacteriosis, bacillary preparations of two strains of bacteria of the genus Bacillus, Bacillus subtilis and Bacillus licheniformis, are offered. It is believed that bacteria of the Bacillus subtilis species are strict aerobes, while representatives of the Bacillus licheniformis species can grow under microaerophilic conditions.

It is also believed that these properties of the two species enhance the effectiveness of probiotic therapy [2-4]. The vast majority of representatives of the genus Bacillus are harmless to warm-blooded animals, exhibit antagonistic activity to a wide range of pathogenic and conditionally pathogenic microorganisms, resistant to lytic enzymes, technologically advanced in production, stable during storage, and environmentally safe.

Considering the possibility of using antibiotics in feed production, it is very promising to use strains with probiotic preparations that have both high antagonistic activity against enteropathogens and multi-resistance to antibiotics.

Known strain of Bacillus subtilis, resistant to rifampicin, ampicillin and streptomycin, with antibacterial activity against pathogenic microorganisms, selected as a prototype [10].

The objective of the present invention is to obtain a strain of Bacillus subtilis with antagonistic activity to a wide range of pathogenic test organisms, as well as resistance to the most widely used antibiotics.

The technical effect that can be obtained by using the obtained strain is to increase the effectiveness of treatment due to the possibility of combined use of a bacterial preparation with antibiotics.

To solve this problem, a strain of Bacillus subtilis subsp.subtilis BKM B-2711D is proposed. The strain is deposited in the All-Russian collection of microorganisms.

Identification of the strain was carried out according to “Identifier of microorganisms” Bergi (1997).

The original soil isolate was isolated in the Kirov region. The soil isolate was mutagenized with nitrosoguanidine (dose from 40 to 100 μg / ml), followed by screening on selective media.

Cultural, morphological and microscopic features of the strain: straight rod-shaped cells, size: 0.5-0.7 × 2.0-3.0 μm, form heat-resistant endospores, not more than 1 in the sporangia cell, spore formation is not suppressed by exposure to air, central location. The cells are motionless. On standard solid nutrient media (MPA, LB, GRM-1) forms small white crater-like colonies. Colonies grow into agar. Catalase: positive test, facultative anaerobic. Used carbon sources for aerobic growth: glucose, arabinose, xylose, beckon, dextrose, casein, starch. The optimum pH for growth is 6.0-8.0, temperature: from + 4 ° C to + 45 ° C.

The invention is illustrated by the following examples.

Example 1. The antagonistic activity of the strain of Bacillus subtilis subsp.subtilis BKM B-271 ID was checked by the method of delayed antagonism.

For this, a suspension of cells washed from the agar medium was seeded with a stroke along the diameter of a Petri dish with agarized Gauze N 2 medium. Crops were incubated in a thermostat at 37 ° C for 48 h. Then, test microorganisms (500 million suspensions) were streaked onto the grown culture. diurnal cultures in physiological saline). Analysis was carried out after 18-20 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 controlled by their parallel growth on plates with agarized Gauze medium No. 2 without the studied culture. The results are shown in the table.

Growth retardation of test cultures, mm E.coli S.typhi-murium S.typhi Ps.aeruginosa St. aureus L.monocytogenes 6 0 9 0 fifteen fifteen

Example 2. Resistance to the following antibiotics was studied: kanamycin, streptomycin, gentamicin, trimethoprim, nalidixic acid, cefazolin, ampicillin, benzylpenicillin sodium salt. The test was carried out during deep cultivation of the producer on a glucose-peptone medium with yeast extract at various concentrations of antibiotics. It was found that the strain has antibiotic resistance: streptomycin -10 μg / ml, tetracycline -10 μg / ml.

Example 3

The enzyme activity in the culture fluid was determined by deep cultivation of the producer on a glucose-peptone medium with yeast extract for two days. The following enzymatic activity values were determined:

α-amylase activity - 0.054 U / ml of medium at pH 6.0,

xylanase activity - 0.18 U / ml of medium at pH 5.0,

protease activity — 2,354 U / ml of medium at pH 7.0.

It should be noted the high protease activity of the strain Bacillus subtilis subsp.subtilis BKM B-2711D.

Literature

1. Smirnov V.V., Reznik S.R., Vyunitskaya V.O. Modern views on the mechanisms of the therapeutic and prophylactic action of probiotics from bacteria of the genus Bacillus // Microbiol. journal - 1993. - No. 4. - S. 92-112.

2. Bakulina L.F., Timofeev I.V., Perminova N.G., Polushkina A.F., Pechurkina N.I. Probiotics based on spore-forming microorganisms of the genus Bacillus and their use in veterinary medicine // Biotechnology. 2001, No. 2, S. 48-56.

3. Hong, N.A., Due L.H., Cutting S.M. The use of bacterial spore formers as probiotics // FEMS Microbiol. Rev. - 2005. - Vol.29, N 4. - P.813-835.

4. Probiotics Microbes: The Scientific Basis / R.J. Collier, et al. // A Report from American Academy of Microbiology - Washington, 2006 .-- 28 p.

5. RF patent 2130316. Therapeutic and prophylactic biological product bactisporin. 1999.

6. USSR patent 1710575. The bacterial strain Bacillus sp. - a component of a therapeutic and prophylactic drug against dysbiosis and allergies. 1992.

7. RF patent 2035186. Preventive biological product sporolact. 1995.

8. Aleshkin, A.V., Afanasyev, S.S., Davydkin, V.Yu., Manko, T.N., Davydkin, I.Yu. Bacterial drug of the new generation "Sporobacterin" // Honey. file cabinet. - 2003. - No. 4.

9. RF patent No. 2035185 Preventive biological product Subalin. - 1995.

10. RF patent 2115725. The strain Bacillus subtilis, resistant to rifampicin, ampicillin and streptomycin, with antibacterial activity against pathogenic species of microorganisms. 1998.

Claims (1)

The strain Bacillus subtilis subsp.subtilis BKM B-2711D, which has a pronounced antagonism against Escherichia coli, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes and resistance to streptomycin and tetracycline.