Amycolatopsis fructiferi subsp ristomycini strain - producer of antibiotic ristomycin

FIELD: medicine.

SUBSTANCE: Amycolatopsis fructiferi subsp. ristomycini 8765 strain comes from mutant transformation of Proactinomyces fructiferi var. ristomycini 5339 strain properties as a result of consequent three-step actions on the stock strain by methylnitrosoguanidine, ultraviolet and gamma-radiation of 60Co with following selection of mutated strain possessing increased antibiotic activity. Obtained strain differs from already known one with growth form on diagnostic agar media, carbon sources assimilation rate (saccharose, starch, rhamnose, and mannose) chromogenic ability and antibiotic activity which is 5 times higher of Proactinomyces fructiferi var. ristomycini 5339 strain activity and is equal to 2000 mcg/ml. Strain is deposited to All Russian Collection of Industrial Microorganisms, collection # S1085.

EFFECT: invention can be used in medical industry for antibacterial antibiotic ristomycin manufacture; ristomycin is effective in treatment of infections induced by gram-positive microorganisms.

1 ex

 

The invention relates to the medical industry and relates to a new strain - producer of antibacterial antibiotic ristomycin from the group of polycyclic glycopeptides (delegation).

The structure of antibiotic ristomycin

With the unique ability to inhibit the biosynthesis of the peptidoglycan of the cell wall antibiotics of this group is effective against many pathogenic gram-positive bacteria. Like vancomycin and teicoplanin, ristomycin a strong antibacterial effect on staphylococci, enterococci and pneumococci, including methicillinsensitive and some anaerobes causing severe septic diseases. Ristomycin used as diagnosticum in some diseases of the blood, and is part of the veterinary drugs.

A known strain Proactinomyces fructiferi var. ristomycini 5339 - producer of antibiotic ristomycin [1], adopted as a prototype. The disadvantage of this strain is the low levels of the antibiotic in the culture fluid (not more than 400 mg of ristomycin on 1 l of culture fluid).

The essence of the invention lies in the fact that the new strain - producer of ristomycin - in a multistage mutagenic effects methylnitrosourea, ultraviolet and the Amma-ray 60With the original strain, followed by selection of mutants on media containing streptomycin and vancomycin as selective agents.

The new strain is deposited in culture collections "Gosniigenetika" under the strain VKPM-S1085.

Strain Amycolatopsis fructiferi subsp. ristomycini PMBC-S1085 has increased productivity of ristomycin (2.0 g in 1 l of culture fluid) and is characterized by the following cultural-morphological and physiological traits.

Cultural and morphological characteristics of the strain VKPM-S1085

Temperature 37°C; duration of cultivation - (7-12) days. Inoculation on agar medium spore material (8-10)-day culture grown on pea agar. Color is defined according to table Bondartsev and Prauser (table 1).

Morphological features. Shporonosni direct, spores oblong, surface spores smooth. Vegetative hyphae septate mycelium. Split into fragments (2-3 µm in length).

Physiological and biochemical properties. Aerobe grows at (24-37)°C; optimum growth at 37°C, biosynthesis 28°C. Gelatine thins, peptonizing milk. Sucrose inverts. Starch is not hydrolyses. On the tissue does not grow. Restores nitrates to nitrites. Melanoidin pigments are not formed during growth on the environment Tresner with citrate of iron.

The use of carbon sources. Well utilisera the t glucose, glycerol, arabinose, maltose; moderately sucrose, starch, mannitol, lactose; poorly - rhamnose, not using xylose.

Antagonistic properties

When tested by the method of the stroke on glucose-nitrate agar No. 2 Gause strain inhibits the growth of gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, Bacillus mycoides, Micrococcus luteus, no effect on Escherichia coli and the yeast Candida albicans. Specific virulent phages have been identified, lysis in the fermentation process does not occur.

Producing strains of ristomycin is illustrated by the following example.

Example 1. Spore material with mycelium culture Amycolatopsis fructiferi subsp. ristomycini PMBC-S1085 grown within (7-10) days at 37°C on pea agar, seeded with a culture medium (100 ml Erlenmeyer flasks with a volume of 750 ml) and grown at 28°C for (18-24) h on a rocking chair at (220-240) rpm

The composition of the seed medium, %:

Glucose1,0
Peptone0,5
The broth of Hottinger3,0
Sodium chloride0,5
WaterWater
pH before sterilization 6,8-7,0

The medium is sterilized for 30 min at a pressure of 0.6 ATM at 120°C.

Grown mycelium in number (3-5) % of the volume of medium is seeded with a culture medium (100 ml Erlenmeyer flasks with a volume of 750 ml) of the following composition, %:

Glycerin2,0
Soy flour0,5
Sodium chloride0,3
Chalk0,5
WaterWater
pH before sterilization6,8

The fermentation is carried out at 28°C for (120-144) h on a rocking chair at (220-240) rpm Antibiotic activity in the culture fluid is determined by the method of diffusion in agar with the use of test-cultures of Bacillus subtilis ATCC 6633. The content of ristomycin in the culture fluid to the end of fermentation is 2000 ug/ml

The drug is derived from a new producer strain by adsorption on ion-exchange resins and multi-stage chemical cleaning [2]due to their physical-chemical characteristics, antibacterial activity and toxicity fully with esthet standard sample of ristomycin.

An advantage of the claimed strain compared with the prototype is the ability to biosynthesis with high productivity glycopeptides antibiotic ristomycin.

Thus, created a new mutant strain of Amycolatopsis fructiferi subsp. ristomycini PMBC-S1085, differing from the prototype by the nature of the growth diagnostic agar media, degree of assimilation of the carbon source (rhamnose, sucrose, starch, mannose)in the ability of education pigment and antibiotic activity, which is 5 times higher than the activity of a strain of the prototype and is 2000 ug/ml (Tables 2 and 3).

LITERATURE

1. Gavrilin, GV, Rokhlenko YEAR Education of ristomycin on synthetic media. In Proc. "Ristomycin and its application in the clinic", Publishing house "Medicine", Moscow, 1965, p.7-13.

2. Brazhnikov MG, Lomakina N.N., Lavrov F, Yurina MS and Klyuyev L. isolation and properties of ristomycin // Antibiotics, No. 5, s (1963).

Table 1
Cultural and morphological characteristics of strain Amycolatopsis fructiferi subsp. ristomycini PMBC-S1085
Agar mediumGrowth aerial mycelium*The color of the myceliumThe solution is the range of the pigment
AirSubstrate
No. 1 Gause+-Yellowish-white, Sooa, poorBrownish (D-3)No
Oatmeal+White (D-3) to pale cream (B6)ColorlessNo
Of čapek+White (D-3)Light brown (D-4)-
Glucose-aspartic+White (D-3)Yellowish (Soaw)-
Lindenbaum (glycerol-nitrate)+++White (D-3) to light-sand (Sooa)Light brown (D-4)
Glucose-nitrate
No. 2 Gause
++White (D-3)Yellowish (Soaw)-
Tresner (peptone and iron citrate) +-White (D-3)ColorlessNo
*/++ moderate;+, weak.

Table 2
Comparative cultural and morphological properties of a new strain of Amycolatopsis fructiferi subsp. ristomycini PMBC-S1085 and strain-prototype 5339
Agar mediumThe growth of the mycelium and the pigment
A new strainStrain-prototype
No. 1 GauseScarce, substrate mycelium brownish B3Moderate, substrate mycelium reddish B1
No. 2 GauseScarce, substrate mycelium white D3Moderate, substrate mycelium brownish B3, a water soluble pigment
Of čapek with sucroseGood aerial mycelium white D3 to sand SooaModerate aerial mycelium pinkish 4B, substrate mycelium orange-brown B1, p is gment lilac

Table 3
Comparative cultural and morphological properties of a new strain of Amycolatopsis fructiferi subsp. ristomycini PMBC-S1085 and strain-prototype 5339
Agar mediumThe growth of the mycelium and the pigment
A new strainStrain-prototype
Assimilation sources
carbon:
SucroseModerateGood
StarchModerateGood
The rhamnoseWeakNot utilize
MannitolGoodWeak
Water-soluble pigmentNoForms
EN Biotechnica activity 2000400
mg/ml
The range of variability1000-3000200-600
activity, ug/ml

The applicant, patentee:

GU research Institute to find new antibiotics to them. Grause Russian Academy of medical Sciences (SU NIINA them. Grause RAMS)

Strain Amycolatopsis fructiferi subsp. ristomycini PMBC-S1085 - producer of antibiotic ristomycin.



 

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