RU2572341C2 - ANTIBIOTIC INA 5812, STRAIN-PRODUCER Streptomyces roseoflavus INA-As-5812 AND METHOD FOR ANTIBIOTIC OBTAINING - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to field of biochemistry, in particular to biologically active compounds, which demonstrate antibacterial activity. Claimed is natural glycopeptide antibiotic INA 5812, which can be applied as medication in medical practice. The invention also relates to strain-producer Streptomyces roseoflavus INA-As-5812, which forms antibiotic INA 5812, and method for obtaining antibiotic INA 5812.

EFFECT: invention makes it possible to obtain novel antibacterial medication with high indices of antibacterial and chemotherapeutical activity and low values of acute toxicity.

3 cl, 4 tbl

Description

The invention relates to biologically active compounds exhibiting antibacterial activity, namely the antibiotic INA 5812, which can be used as a medicine in medical practice.

The proposed compound, producer strain and method for producing an antibiotic are not described in the patent and scientific literature.

The proposed natural compound, the antibiotic INA 5812, refers to a group of glycopeptide compounds characterized by a peptide structure of amino acid chains and the presence of a sugar residue.

Antibiotics of this group have activity against gram-positive bacteria and mycobacteria, including Mycobacterium tuberculosis. The advantage of glycopeptide antibiotics over a number of antibiotics of other groups is the bactericidal type of action and high activity against microorganisms resistant to many drugs, including β-lactam antibiotics and fluoroquinolones. The search for new drugs of a glycopeptide structure, less toxic and more active, is promising among the biosynthesis products of actinomycete cultures. Among these natural glycopeptide compounds, antibacterial antibiotics vancomycin, teicoplanin and ramaplanin are currently used in medical practice, effective for severe purulent-septic diseases caused by gram-positive microorganisms, especially staphylococci, including methicillin-resistant strains (MRSA), enterococcus, sepsis, as well as membrane colitis due to their high activity against the anaerobic pathogen Clostridium difficile.

The aim of the invention is the creation of a natural glycopeptide antibiotic with antibacterial activity and expanding the arsenal of means of influence on gram-positive bacteria.

The goal is achieved by isolating the soil strain of Streptomyces roseoflavus INA-Ac-5812 producer of the new glycopeptide antibiotic INA 5812, which has high antibacterial activity. The antibiotic INA 5812, formed during the cultivation of a new strain INA-Ac-5812, has a mol. mass (MALDI-TOF-MS method) 1917.8 Yes.

The antibiotic gives positive reactions to free amino groups (with ninhydrin), to carbohydrates (Molish and Feling reactions), to phenolic hydroxyls (with FeCl ₃ and Pauli reaction). It has strong blue fluorescence in UV light and a characteristic UV spectrum with λ _max (methanol) at 237, 262 and 360 nm. The IR spectrum has strong absorption bands in the region: ν _max , cm ^-1 : 3301; 3062; 2927; 1645; 1538; 1382; 1231; 1072; 1003; 702.

A characteristic distinguishing feature of the antibiotic INA 5812 as a glycopeptide compound is the amino acid composition. The following amino acids were found in the antibiotic acid hydrolyzate: β-alanine, glycine, ornithine, tyrosine, α, β-diaminobutyric acid, alanine, proline, leucine, aspartic acid, series and three non-protein amino acids. The antibiotic contains neutral sugar arabinose.

The essence of the invention lies in the fact that a new producer strain Streptomyces roseoflavus was obtained as a result of a systematic screening of soil cultures of streptomycetes forming antibacterial glycopeptide antibiotics.

The resulting new strain was deposited in the culture collection of FSBI NIINA named after G.F. Gauze RAMS under the number INA-As-5812.

Strain INA-Ac-5812 accumulates an antibiotic in the culture fluid (2-5 μg / ml) and is characterized by the following cultural, morphological and physiological characteristics.

Cultural, morphological and physiological characteristics of the strain INA-Ac-5812

Temperature (28-37) ° C; growing time (7-21) days. Spore seed of a ten-day culture of the strain grown on Lindenbein glycerin nitrate agar medium. The color is determined according to the table of Bondartsev and Prauser (Table 1).

Morphological signs. The spore chains are straight, convoluted, the surface of the spores is smooth.

Physiological and biochemical properties. Aerobe. Grows at (24-37) ° C, optimum growth 28 ° C and in the range of pH values (5.0-10.0) with optimum growth at pH 7.0; gelatin dilutes. Milk peptones. Sucrose inverts. Starch hydrolyzes. Fiber does not grow. Restores nitrates to nitrites. It does not form melanoid pigments when growing on Tresner's medium with peptone and ferric citrate. Attitude towards carbohydrates: makes good use of fructose, maltose, rhamnose; moderately glucose, arabinose, lactose; poorly uses sucrose, sorbitol, mannitol; does not use raffinose and cellulose when grown on media with an inorganic nitrogen source.

Storage. The strain is stored in the form of spores on sterile soil with silica sand or on millet grains, as well as a mature culture on agar media under liquid paraffin.

Antagonistic properties. When tested by the bar method on glucose-nitrate agar No. 2, the Gauze strain inhibits the growth of gram-positive bacteria and yeast-like fungi. The strain is phage resistant. Phage in culture is absent.

The invention is illustrated by the following example.

Example. To obtain spore material, a culture of Streptomyces roseoflavus strain INA-Ac-5812 is grown for (7-10) days at 37 ° C on agar medium No. 2 of Gouse of the following composition,%:

Tripton 0.3 Glucose 1,0 Peptone 0.5 Agar 3.0 NaCl 0.5 Water Tap pH before sterilization 7.0-7.2

The medium is sterilized for 30 minutes at a pressure of 1.0 atm and a temperature of 120 ° C.

To obtain seed mycelium, culture spores are sown on a nutrient medium of the following composition,%:

Hottinger Broth (28-30 mg% amine nitrogen) 3.0 Peptone 0.5 Glucose 1,0 Calcium carbonate 0.2 Sodium Chloride 0.5 Water Tap pH before sterilization 7.0-7.2

The seed is grown at 28 ° C for 48 hours in 750 ml Erlenmeyer flasks; the amount of medium in the flasks is 150 ml. Rocking rotation speed (200-240) rpm The amount of seed for inoculation is (5-7)%.

Fermentation is carried out on a shaker at 28 ° C on medium (100 ml in Erlenmeyer flasks with a capacity of 750 ml) of the following composition,%:

Soya flour 3.0 Glucose 3.0 NaCl 0.3 CaCO ₃ 0.3 Water Tap pH before sterilization 7.0-7.2

The antibiotic accumulates in the producer fluid.

After 120 hours of fermentation, the antibiotic activity of the culture fluid is determined by agar diffusion using a Bacillus subtilis 6633 ATCC test microorganism.

To isolate the antibiotic, a native solution (culture fluid filtered from mycelium) with a volume of 5 l, whose pH (6.8-7.3), is passed through a column of 500 ml of Amberlit XAD-2 resin (Serva, Germany) at a speed of 120 ml / cm ² h At the end of sorption, the column is washed with distilled water (1 L). The antibiotic INA 5812 was eluted from the resin with a mixture of acetone-n-butanol-water (1: 1: 1) at pH 3.0. The organic solvents of the eluate were evaporated in vacuo and the antibiotic was extracted from the aqueous residue with n-butanol and sorbed for further purification on a column with DEAE-Sephadex (in OH ^- form). The active fractions are combined, the solvent is removed in vacuo, and the antibiotic is precipitated from 50% aqueous-alcoholic solution with acetone. The obtained antibiotic raw materials are chromatographed on a C-18 reverse phase column using a gradient of acetonitrile in water and a pure antibiotic is obtained in the form of a white powder, readily soluble in water, methanol, to a limited extent in ethanol and insoluble in other organic solvents. From 5 l of the native solution, 10 mg of the substance are obtained. The activity of the drug against Bacillus subtilis 6633 ATCC is 3000 units. dilutions / mg.

Thus, a new strain of Streptomyces roseoflavus INA-Ac-5812, the producer of the new glycopeptide antibiotic INA 5812, was obtained and a method of biosynthesis, isolation and chemical purification was proposed, which allows one to obtain a biologically active compound - the antibacterial antibiotic INA 5812.

The new drug - the antibiotic INA 5812 - has high antibacterial and chemotherapeutic activity and low acute toxicity.

The IPC values (minimum inhibitory concentration) determined in vitro for two standard staphylococcus strains are presented in Table 2.

Indicators of chemotherapeutic activity in vivo obtained on a model of staphylococcal sepsis of white mice are presented in Table 3.

The results show the high effectiveness of the INA 5812 antibiotic as a drug: a dose of 2.5 μg / kg provides 100% cure for staphylococcal sepsis.

Acute antibiotic toxicity was determined on white mice and the results are shown in Table 4. The maximum tolerated dose of the INA 5812 antibiotic is ten times higher than the dose of 100% cure for mice from staphylococcal sepsis.

Table 1 Cultural and morphological characteristics of the strain Streptomyces roseoflavus INA-Ac-5812 Agar medium Aerial mycelium growth ^* Mycelium color Soluble pigment Air Substrate No. 1 Gause +++ Grayish-white to pinkish (D-3, R-2) Pale brownish (B-4) Light yellow (D-4) Oatmeal ++ Pale brownish (B-6) Grayish-yellow (D-3, Zh-1) No Capek +++ Whitish (D-3) Brownish-yellowish (D-4) No Glucose Aspartic + Yellowish (F-1) Pale brownish (B-4) No Lindenbein (glycerol nitrate) +++ Whitish cream (D-3, Coo6) Yellowish Cream (B-6) No Krasilnikova SR-1 No growth No. 2 Gause ++ Whitish-pinkish (D-3, G-3) Whitish-creamy (D-3, B-6) No Tresner (with peptone and citric acid iron) Melanoid pigments do not form * / (+++) good; (++) - moderate; (+) - weak.

table 2 Antistaphylococcal activity in vitro of the antibiotic INA 5812 Staphylococcus strain No. The minimum inhibitory concentration (MIC), μg / ml St. aureus 25923 ATCC 0.5 St. epidermis 12228 ATCC 0.5

Table 3 The effectiveness of the antibiotic INA 5812 in the treatment of staphylococcal sepsis in white mice Staphylococcus strain used for infection The dose of the drug (mg / kg), protecting against death 50% of infected animals (ED ₅₀ ) ^* 100% infected animals Clinical isolate, No. 10 0.37 ± 0.05 2.5 ± 0.07 * / ED ₅₀ (50% lethal dose) according to the method of accumulation of Behrens frequencies.

Table 4 Acute toxicity of the antibiotic INA 5812 after intravenous administration in in vivo experiments Toxicity indicator Dose, mg / kg animal weight LD50 (Lidfield and Wilcoxon method) 53.6 (ref. Value 41.9-65.3) MTD (maximum tolerated dose) 28.5 ± 0.5

Claims (3)

1. The strain Streptomyces roseoflavus INA-AC-5812-producing antibiotic. 2. A method of producing an INA 5812 antibiotic, which involves culturing the producer of Streptomyces roseoflavus INA-Ac-5812, sorption of the antibiotic from the native solution on Amberlite XAD-2 (Serva, Germany), followed by its desorption with acetone-n-butanol-water mixture (1: 1: 1) and isolation of the antibiotic by extraction with n-butanol, sorption of the antibiotic on DEAE-Sephadex, desorption of 0.05N. CH ₃ COOH in 50% ethanol, concentration, precipitation from 50% aqueous-alcoholic solution with acetone and final purification of the crude preparation on a C-18 reverse phase column in the form of a white powder, readily soluble in water and methanol. 3. The new natural glycopeptide antibiotic INA 5812, obtained by the method according to p. 2, mol. mass of 1917.8 Da, with a characteristic UV spectrum with λ _max (methanol) at 237, 262 and 360 nm and an IR spectrum in the region: v _max : 3301; 3062; 2927; 1645; 1538; 1382; 1231; 1072; 1003 and 702 cm ^-1 containing a specific set of amino acids - β-alanine, glycine, ornithine, tyrosine, α, β-diaminobutyric acid, alanine, proline, leucine, aspartic acid, serine, three non-protein amino acids and neutral sugar arabinose and having high indicators of antibacterial and chemotherapeutic activity in the treatment of staphylococcal sepsis in white mice in in vivo experiments.