RU2405821C1 - 'pashkov' bacillus pumilus bacteria strain producer of biologically active substances exhibiting antagonistic activity on opportunistic, pathogenic bacteria, yeast fungi and viruses - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention can be used for making a probiotic preparation exhibiting an antagonistic effect on opportunistic and pathogenic bacteria and yeast fungi, as well as expressed anti-enteroviral activity. 'Pashkov' Bacillus pumilus bacteria strain is recovered from the environment in the Department of Microbiology, Virology and Immunology of the I.M.Sechenov Moscow Medical Academy, and deposited in the L.A.Tarasevich State Standartisation and Control under No. 286.

EFFECT: strain shows a wide spectrum of antagonistic activity, low adhesive activity, high anti-enteroviral activity of strain secretion products.

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Description

The invention relates to biotechnology and can be used in the microbiological industry to obtain a probiotic preparation that has an antagonistic effect on opportunistic and pathogenic bacteria and yeast fungi, as well as a pronounced antiviral activity against enteroviruses.

The strains exhibiting high antagonistic properties against a number of bacteria and fungi are widely known.

The known strain of Bacillus subtilis 534 is the producer of the probiotic Sporo-bacterin, which is intended for the prevention and treatment of the gastrointestinal tract, dysbiosis (SU 1708350, class A61K 35/66).

The disadvantages of this strain are the short shelf life, because it contains live bacteria that cannot retain their properties for a long time. Due to its low purity, the drug has a narrow scope as a feed additive for animals. The strain is also sensitive to antibiotics, which limits the scope of the drug.

A known strain of Bacillus subtilis 3H (GISK 248), which has the property of antibiotic resistance, is used to obtain the probiotic drug "Baktisporin", which is used together with antibiotics for the treatment and prevention of dysbiosis, digestive enzyme deficiency, purulent infections, food allergies (RU 2067616 C1, cl A61K 35/74, 1996).

A known bacterial strain Bacillus subtilis 1719 is a producer of antagonistically active biomass against pathogens, as well as proteolytic, amylolytic and lipolytic enzymes (RU 2298032 C2, class C12N 1/20, C12N 9/14, A61K 35/74, 2007).

The disadvantage of these strains is the lack of their antiviral activity.

The problem to which the invention is directed, is to obtain a new strain - a producer of antagonistically active biomass against pathogenic microbes, including viruses.

To solve this problem, a bacterial strain Bacillus pumilus "Pashkov" is proposed, deposited by the Collection of the State Research Institute of Standardization and Control of Medical Biological Preparations named after L.A. Tarasovich under the number 286.

The bacterial strain Bacillus pumilus "Pashkov" isolated from the environment at the Department of Microbiology, Virology and Immunology MMA named after I.M.Sechenova. The strain exhibits a wide range of antagonistic activity, low adhesive activity.

Characteristics of the strain.

Cultural, morphological and other properties of the strain. The cells of the strain are large gram-positive aerobic bacilli with centrally or paracentrically located spores of ellipsoid or cylindrical shape, accumulations of cells in the form of chains can occur. When studying the colonies of the strain in oblique light, it was found that the colonies are granular with a prominent dense center, the edges are uneven. Microscopy revealed that the cells are large, barrel-shaped sticks, sometimes connected in chains. Depending on the cultivation conditions and culture medium, the colonies can form a pigment from beige to pink. The strain has low adhesiveness. The strain is harmless, non-toxic, avirulent.

Growth on solid nutrient media. The strain of bacteria Bacillus pumilus "Pashkov" grows on simple nutrient media. Film growth is observed on the surface of the agar.

Growth on liquid nutrient media. The strain of bacteria Bacillus pumilus "Pashkov" grows on the surface of liquid nutrient media.

Biochemical activity of the strain. The bacterial strain Bacillus pumilus Pashkov is able to utilize glucose, xylose, beckon, sucrose and maltose. Does not utilize lactose and salicin. Does not form gas during glucose breakdown. Hydrolyzes casein and does not hydrolyze starch. Does not form gas during glucose breakdown. Does not restore nitrates to nitrites, does not produce indole, hydrogen sulfide. Provides urease. Causes a liquefaction of 10% gelatin. It does not have hemolytic properties and lecithinase activity.

Homologous phages to the strain were not identified.

The ratio of strain to species diagnostic sera. Diagnostic sera are not available.

Genetic features of the strain (auxotrophy, antibiotic resistance, etc.). The bacterial strain Bacillus pumilus Pashkov does not have plasmids, is resistant to oxacillin, ampicillin, cefuroxime, cefotaxime, ceftriaxone, erythromycin, tetracycline, clindamycin, levomycin, rifampicin, bacitracin, fucidocin, fuzidocin, fuzidocin, fuzidocin, fuzidocin, fuzidocin, fuzidocin, fuzidocin.

The activity (productivity) of the strain, as well as other production indicators of the strain. The product synthesized by the strain is biologically active substances that have an antagonistic effect on opportunistic and pathogenic bacteria and yeast fungi, as well as having antiviral activity.

The activity of the strain is evaluated by determining the delayed antagonism or by co-cultivation with test strains. The inhibitory properties of the culture fluid of the bacterial strain Bacillus pumilus “Pashkov” is evaluated by the effect on the growth kinetics of test cultures at an initial concentration of 10 ⁶ CFU / ml using a microbiological analyzer with a tablet photometer BIOSCREEN / iEMS - reader MF, LabMetod, Finland. The bacterial strain Bacillus pumilus "Pashkov" is a producer of biologically active substances that have antagonistic properties against pathogenic microbes. It is promising for the creation of probiotic drugs and drugs against enterovirus infections.

Conditions and composition of storage media. The bacterial strain Bacillus pumilus "Pashkov" is stored on semi-liquid agar frozen at T = -70 ° C for 1 year.

The conditions and composition of the media for the propagation of the strain. The strain of bacteria Bacillus pumilus "Pashkov" is propagated by plating on liquid and agarized nutrient media (meat and peptone broth, meat and peptone agar).

The conditions and composition of the media for the production of toxin, antigenic fractions, pigment and other cellular waste products. The bacterial strain Bacillus pumilus "Pashkov" does not produce toxins. On agarized media, the colonies of the strain form a pigment from beige to pink.

Example. The bacterial strain Bacillus pumilus "Pashkov" isolated from the environment at the Department of Microbiology, Virology and Immunology MMA named after I.M.Sechenova. The strain is antagonistically active against opportunistic and pathogenic bacteria: Staphylococcus, Proteus, Shigella, Escherichia, Pseudomonas and fungi of the genus Candida, Rhodotorula, Debaryomyces. The study of the adhesive properties on human erythrocytes (group 0 Rh +) showed a low adhesive activity of the strain presented for deposition (adhesion index 1.76 ± 2.5). In animal experiments, the strain was harmless, non-toxic, avirulent. Plasmids in the studied strain were not found.

A wide spectrum of antagonistic activity, a low level of adhesive activity, and the production of highly active biological substances characterize the bacterial strain Bacillus pumilus "Pashkov" as promising for the creation of probiotic preparations on its basis and its use as a producer of biologically active substances with antibacterial, antifungal and antiviral activity.

Determination of antibiotic resistance and plasmid profile of B. pumilus Pashkov strains is one of the important properties that determine the possibility of its use in conjunction with antibacterial drugs. In this regard, the antibiotic resistance of B. pumilus Pashkov strains was studied in relation to 23 antibiotics. Strain B. pumilus "Pashkov" was resistant to oxacillin, ampicillin, cefuroxime. cefotaxime, ceftriaxone, erythromycin, tetracycline, clindamycin, levomycetin, rifampicin, bacitracin, fusidine, novobiocin. It is sensitive to cephalexin, trimethoprim with sulfamethoxazole, amikacin. The data obtained are presented in table 1.

When studying the plasmid profile of bacteria B. pumilus "Pashkov" plasmids were not detected.

The influence of the B. pumilus Pashkov strain on the macroorganism was evaluated by the toxicity, toxigenicity, virulence, and safety of the B. pumilus Pashkov strain in in vivo experiments. From table 2. it is seen that the strain B. pumilus "Pashkov" did not possess virulence in the tested concentrations. All animals remained alive during the observation period (5 days), none of them showed signs of the disease.

Table 3 presents the results of the study of the toxicity of B. pumilus "Pashkov". The results obtained showed the absence of toxicity in the studied strain.

Table 4 presents the results of determining the toxigenicity of the strain B. pumilus "Pashkov". The results of the experiment indicate the absence of toxigenicity in the studied strain.

In order to more fully study the safety of the strain, increasing doses of the strain were tested when administered orally to mice (Table 5). It was established that a single per os administration of large doses (20 × 10 ⁹ mk / ml) of B. pumilus “Pashkov” led to insignificant weight loss (or the weight did not change) of animals on the first day after administration, on the following days, the weight of animals was growing. In the groups treated with 0.9% NaCl, no death was observed for all methods of administration.

Thus, the studies showed that the B. pumilus Pashkov strain is non-toxic, non-toxic and avirulent, i.e. safe.

The main provisions of the safety manual for companies involved in the production of spore-forming bacteria as probiotics include the requirement of identification to the species using a modern and validated methodology, in particular the study of the sequence of 16 rRNA genes. The data of ribotyping of the 16S subunit of B. pumilus Pashkov strain are presented:

TGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTC

GAGCGGACAGAAGGGAGCTTGCTCCCGGATGTTAGCGGCGGACGGGTG

AGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAA

CCGGAGCTAATACCGGATAGTTCCTTGAACCGCATGGTTCAAGGATGA

AAGACGGTTTCGGCTGTCACTTACAGATGGACCCGCGGCGCATTAGCT

AGTTGGTGGGGTAATGGCTCACCAAGGCGACGATGCGTAGCCGACCTG

AGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC

GGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGA

GCAACGCCGCGTGAGTGATGAAGGTTTTGGGATCGTAAAGCTCTGTTGT

TAGGGAAGAACAAGTGCGAGAGTAACTGCTCGCACCTTGACGGTACCT

AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGT

AGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGC

GGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGAGGGTC

ATTGGAAACTGGGAAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCAC

GTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAA

GGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGA

GCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTG

CTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAA

GCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATT

GACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAA

CGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGAT

AGGGCTTTCCCTTCGGGGACAGAGTGACAGGTGGTGCATGGTTGTCGTC

AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC

TTGATCTTAGTTGCCAGCATTTAGTTGGGCACTCTAAGGTGACTGCCGG

TGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTT

ATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCTGCG

AGACCGCAAGGTTTAGCCAATCCCATAAATCTGTTCTCAGTTCGGATCG

CAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGA

TCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGT

CACACCACGAGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCTTTAT

GGAGCCAG

In order to study the antagonistic properties of biologically active substances produced by the strain, periodic cultivation was carried out on a liquid nutrient medium developed in the laboratory. It was found that bacteria synthesize antimicrobial substances with a wide spectrum of action against bacteria, fungi and viruses (Tables 6, 7, 8).

The table shows that the culture fluid obtained by cultivating the strain B. pumilus "Pashkov" in optimally selected conditions contains biologically active substances with a high bacteriostatic effect.

Analysis of the obtained data indicates a high fungistatic effect of the culture fluid of the bacterial strain B. pumilus "Pashkov".

When studying the antiviral effect of the culture fluid of B. pumilus Pashkov strain, a high activity of the secretion products of the strain was established for the studied enteroviruses (Table 8).

Table 1 The sensitivity of the strain B. pumilus "Pashkov" to antibiotics AB group Group representative Loading dose per disc (mcg) The diameter of the zone of growth inhibition (mm) (X ± m) Result Penicillins 1st generation Benzylpenicillin 10 units 12 ± 3.3 Moderately sensitive Penicillins 2 generations Oxacillin 10 8 ± 3.7 Steady Penicillins 3 generations Ampicillin 10 12 ± 1.6 Steady Penicillins 4 generations Carbenicillin 25 15 ± 0.8 Moderately sensitive Cephalexin thirty 18 ± 3.4 Sensitive Cephalosporins 1st generation Cefazolin thirty 15 ± 0.5 Moderately sensitive Cephalosporins 2nd generation Cefuroxime thirty 9 ± 0.8 Steady Cephalosporins 3 Cefotaxime thirty 8 ± 2.2 Steady generation Ceftriaxone thirty 11 ± 2.6 Steady Macrolides Erythromycin fifteen 14 + 2.2 Steady Clarithromycin fifteen 15 ± 1.4 Moderately sensitive Trimethoprim + Sulfamethoxol-Zol 1.25 / 23.75 13 ± 1.4 Sensitive Quinolones Ofloxacin 5 15 + 0.9 Moderately sensitive Ciprofloxacin 5 16 ± 0.8 Moderately sensitive Aminoglycosides
1 generation Kanamycin thirty 15 + 1.7 Moderately sensitive Aminoglycosides 3 generations Amikacin thirty 16 ± 1.7 Sensitive Tetracyclines Tetracycline thirty 14 ± 1.9 Steady Lincosamides Clindamycin 2 9 ± 0 Steady Chloramphenicol Chloramphenicol thirty 8 ± 0.5 Steady Rifampicin Rifampicin 5 10 ± 2.4 Steady Other Bacitracin 10 units 3.7 ± 0.9 Steady Fusidine 10 10 ± 2.9 Steady Novobiocin 5 9 ± 2.4 Steady

table 2 The study of the virulence of the strain B. pumilus "Pashkov" Number of animals The entered dose, (m.k. / ml) Multiplicity of introduction Number of animals sick the fallen survivors Experience Intraperitoneally 10 0.5 mld. one 0 0 10 10 1 mld. one 0 0 10 10 2 mld. one 0 0 10 To 10 0.9% NaCl one 0 0 10

Table 3 The study of the toxicity of B. pumilus strain "Pashkov" Number of animals The entered dose, (m.k. / ml) Multiplicity of introduction Number of animals sick the fallen survivors ABOUT Intraperitoneally 10 1 mld. one 0 0 10 10 2 mld. one 0 0 10 10 3 mld. one 0 0 10 Comte 10 0.9% NaCl one 0 0 10

Table 4 The study of the toxigenicity of the strain B. pumilus "Pashkov" Number of animals The administered dose (ml) Multiplicity of introduction Number of animals sick the fallen survivors Intraperitoneally ABOUT 10 0.5 one 0 0 10 10 1,0 one 0 0 10 10 0.5 medium Gause one 0 0 10

Table 5 Safety study of B. pumilus Pashkov strain Groups Administered dose Number of animals The average weight of the animals, (g) (X ± m) survivors the fallen Before introduction After 1 day In 5 Orally one 5 mld. 10 0 15.4 ± 0.3 15.6 ± 0.2 16 2 10 mld. 10 0 15.6 ± 0.3 15.3 ± 0.3 16 3 20 mld. 10 0 14.8 ± 0.3 14.1 ± 0.3 fifteen

Table 6 Bacteriostatic effect of the culture fluid of B. pumilus Pashkov strain against opportunistic and pathogenic bacteria Researched
material Suppression
growth
microorganisms (%) S.aureus S.aurerus S.xylosus 25 S.flexneri 337 S.sonnei 170 E.coli O 111: H P.aeruginosa P.mirabilis 24a P.vulgaris 177 Culture liquid B. pumilus "Pashkov" one hundred one hundred one hundred one hundred one hundred one hundred one hundred one hundred one hundred

Table 7 Bacteriostatic effect of the culture fluid of the strain B. pumilus "Pashkov" in relation to clinical strains of yeast fungi Test material Inhibition of microorganism growth (%) C.albicans No. 1 C.albicans No. 2 C.haemuloni C.tropicalis Rhodotor ula spp Debaryo myces hansenii Culture fluid B.pumilus "Pashkov" 94 92 91 89 88 92

Table 8 The effectiveness of the therapeutic and prophylactic action in vitro of the culture fluid of the strain B. pumilus "Pashkov" in relation to 4 enteroviruses, depending on the dilution of the drug and the dose of viruses Viruses Observation time Dilution of the drug, providing 100% protective effect Dilution of the drug, providing 100% preventive effect Virus dose in lg TCD _{50 / 0.5 ml} (h) one hundred fifty 25 12.5 one hundred fifty 25 12.5 Polio-1 48 1:80 1: 160 1: 640 1: 640 1:40 1: 160 1: 160 1: 640 72 1:80 1:80 1: 640 1: 640 1:40 1: 160 1: 160 1: 640 E CHO-3 48 1:40 1:40 1: 640 1: 640 0 1: 160 1: 640 1: 640 72 1:40 1:40 1: 160 1: 320 0 1: 160 1: 640 1: 640 E CHO-6 48 1:40 1: 640 1: 640 1: 640 0 1:20 1: 640 1: 640 72 1:40 1: 160 1: 160 1: 640 0 1:20 1: 640 1: 640 Sox B (1-6) 48 1:80 ^* 1:80 ^* 1:40 1:40 0 1:20 1: 640 1: 640 72 1:80 ^* 1:80 ^* 1:40 1:40 0 1:20 1: 320 1: 640 Note: * - partial cell protection

Claims (1)

The strain of bacteria Bacillus pumilus "Pashkov" GISK them. L.A. Tarasevicha No. 286 - producer of biologically active substances with antagonistic activity against opportunistic, pathogenic bacteria, yeast and viruses.