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
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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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The invention relates to the field of biotechnology, namely the probiotic bacterial preparations, which can find application in veterinary medicine.
Probiotics are bacterial preparations on the basis of strains of microorganisms with antagonistic activity against enteropathogenic bacteria.
Classical studies Iaichikova these properties have been studied on the example of lactic acid bacteria. Then circle the producers of probiotic preparations was expanded.
Probiotics based on strains of spore-forming bacteria relatively recently have they been applied in veterinary medicine. Antagonistic properties of these bacteria have attracted the attention of many developers.
The most promising producers are strains with antagonistic activity not only for enteropathogenic bacteria, but also to a wide range of test organisms, which cause extraintestinal infections.
The complexity of the treatment of infectious diseases is the occurrence of pathogenic microorganisms in polyresistance to antibiotics. In addition, the antibiotics often have side effects on the human body and cause dysbiosis, aggravating the course of the underlying disease. This necessitates the search for new approaches to data is th problem.
Drugs probiotics on the basis of bacteria of the genus Bacillus, which suppress the development of pathogenic microorganisms, are widely used in agriculture to suppress phytopathogenic bacteria and fungi in medicine, veterinary medicine for the treatment of various diseases of poultry and farm animals [1].
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. Proposed numerous ways probiotics from sporoobrazuyushchikh bacteria: backspin [5], vacillatory [6], sporulant [7], sporobacterin [8], sublin [9].
To increase the effectiveness of prevention and treatment of dysbacteriosis offer bacillary drugs from two strains of bacteria of the genus Bacillus is Bacillus subtilis and Bacillus licheniformis. It is believed that bacteria of the species Bacillus subtilis - strict aerobes, whereas the species Bacillus licheniformis can grow in microaerophilic conditions.
It is also believed that these properties are of two types strengthen the effectiveness of probiotic therapy [2-4]. The vast majority of members of the genus Bacillus are harmless to warm-blooded animals, exhibit antagonistic activity against a wide spectrum of pathogenic and conditionally pathogenic microorganisms resistant to lytic enzymes, easy to manufacture,stable during storage, environmentally safe.
Given the possibility of the use of antibiotics in feed, is very promising use in the composition of probiotic preparations of strains as high antagonistic activity against enteropathogens and MDR to antibiotics.
Known strain of Bacillus subtilis resistant to rifampicin, ampicillin and streptomycin, with antibacterially activity against pathogenic microorganisms, selected as a prototype [10].
The present invention is to obtain a strain of Bacillus subtilis, which has antagonistic activity to a wide range of pathogenic test organisms, as well as resistance to most commonly 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 bacterial medication with antibiotics.
To solve this problem, a strain of Bacillus subtilis subsp.subtilis BKM B-2711D. The strain deposited at the all-Russian collection of microorganisms.
Identification of the strain was carried out according to the "identification of microorganisms" Berga (1997).
The original soil isolate selected in the Kirov region. Soil isolate was subjected Muta the military impact of nitrosoguanidine (dose from 40 to 100 µg/ml) followed by screening on selective media.
Cultural-morphological and microscopic features of the strain: the cells are rod-shaped, straight, size: 0,5-0,7×2.0 to 3.0 μm, to form heat-resistant endospores, no more than 1 in the cell-sporangia, the sporulation is not inhibited by exposure to air, Central location. Cells are fixed. On standard nutrient dense environments (MPA, LB, RM-1) forms small white crateriform colony. Colonies grow into the agar. Catalase: positive test, optional gone anaerobic. Used carbon sources under aerobic growth: glucose, arabinose, xylose, lures, dextrose, casein, starch. The optimum pH for growth of 6.0-8.0, temperature: +4°C to +45°C.
The invention is illustrated by the following examples.
Example 1. Antagonistic activity of Bacillus subtilis strain subsp.subtilis BKM B-271 ID was checked by the method of deferred antagonism.
For this purpose, a suspension of cells, washed from the agar medium, were sown by the bar diameter Petri dishes with agar medium Gause N 2. Crops were incubated in a thermostat at 37°C for 48 hours and Then grown culture was podseval stroke test microorganisms (500 millionth suspension subsistence crops in saline solution). Analysis was performed after 18-20 hours of incubation at 37°C largest zones of no growth of the test cultures. Control of the growth of the test cultures was parallel cultivation and the Cup with agar medium Gause N 2 without the studied culture. The results are shown in the table.
| The stunted growth of the test cultures, mm | ||||
| E.coli | S.typhi-murium | S.typhi | Ps.aeruginosa | St. aureus | L.monocytogenes |
| 6 | 0 | 9 | 0 | 15 | 15 |
Example 2. We studied the resistance to the following antibiotics: kanamycin, streptomycin, gentamicin, trimethoprim, nalidixic acid, Cefazolin, ampicillin, benzylpenicillin sodium salt. The audit was carried out in submerged cultivation producer on glucose-peptone medium with yeast extract at various concentrations of antibiotics. It was found that the strain has resistance to antibiotics: streptomycin -10 μg/ml, tetracycline -10 mcg/ml
Example 3.
The enzyme activity in the culture fluid was determined in submerged cultivation producer on glucose-peptone medium with yeast extract in two days. The following were identified values of enzyme activity:
α-amylase activity - 0,054 IU/ml among the s at pH of 6.0,
silananda activity of 0.18 U/ml of medium at pH 5.0,
proteasa activity - 2,354 IU/ml of medium at pH 7.0.
Note the high protease activity of Bacillus subtilis strain subsp.subtilis BKM B-2711D.
Literature
1. Smirnov V.V., Reznik S.R., Kunicka V.O. current views on the mechanisms of therapeutic and preventive effect of probiotics bacteria of the genus Bacillus // Microbiol. Journe. - 1993. No. 4. - Pp.92-112.
2. Bakulina L. F., I. Timofeev, N. Perminov, Polushkina A.F., Pechurkin NI Probiotics on the basis of spore-forming microorganisms of the genus Bacillus and their use in veterinary medicine //Biotechnology. 2001, No. 2, P.48-56.
3. Hong, N.A., Due L.H., S.M. Cutting The use of bacterial spore formers as probiotics // FEMS Environ. Rev. - 2005. - Vol.29, N 4. - P.813-835.
4. Probiotics Microbes: The Scientific Basis / R.J. Collier, et al. // A Report from the American Academy of Microbiology Washington, 2006. - 28 p.
5. RF patent 2130316. Therapeutic biological product, backspacing. 1999.
6. Patent USSR 1710575. The bacterial strain Bacillus sp. - component treatment-and-prophylactic drug against dysbacteriosis and allergies. 1992.
7. RF patent 2035186. Preventive biological sporulant. 1995.
8. Aleshkin, A., Afanasiev, S., Davydkin, V.Y., Manko, T.N., Davydkin, IU Bacterial preparation of a new generation "Sporobacterin" // the Honey. card index. - 2003. No. 4.
9. RF patent №2035185 Preventive biological preparation of Sublin. - 1995.
10. RF patent 2115725. The Bacillus subtilis strain resistant is to rifampicin, ampicillin and streptomycin, with antibacterially activity against pathogenic microorganisms. 1998.
The strain of Bacillus subtilis subsp.subtilis BKM B-2711D, with the strong antagonism against Escherichia coli, Salmonella typhi, Staphylococcus aureus, Listeria monocytogenes and resistance to streptomycin and tetracycline.