SU1406156A1 - Strain of aerococcus viridans bacteria for producing clones with oxidase and reductase activity - Google Patents

Abstract

The invention relates to microbiology and relates to the production of KvjTbTyp with an increased ability to jn population and increased biological activity. The purpose of the invention is to obtain a bacterial strain to obtain clones with an increased ability to grow a population and increased oxidase and reductase activity. As a culture for breeding clones with increased biological activity, the Aerococcus strain (Musosos-CUS hyperoxydans) VNIIA-1730 (Collection of microorganisms of the All-Union Antibiotic Research Institute) is used, which produces clones with increased oxidase and reductase activity. 5 tab.

Description

four

Od

SP

ABOUT)

The invention relates to microbiology and relates to the production of a strain that can be used to select clones with increased oxidase and reductase activity.

The aim of the invention is to obtain a bacterial strain for obtaining clones with an increased ability to grow a population and increased oxidase and reductase activity.

The strain Aerococcus viridans (Muso-COCCUS hyperoxydans) VNIIA No. 1730 (GK 308) was obtained by selecting a non-hemolytic colony on a 3% blood-co-peptone agar among the colonies of the natural sifting MusoCCUS hyperoxydans 167VR. From the selected non-hemolytic colony, a pure culture was obtained by replanting it onto m-peptone agar containing 10% horse serum.

The strain Aerococcus viridans (Muso-COCCUS hyperoxydans) GK308 was deposited in the collection of microorganisms of the All-Russian Scientific Research Institute of Antibiotics under number 1730. The strain has characteristic properties.

Morphological signs. Polymorphic. The main structures are cocci of elongated ovoid shape, gram-positive, immobile, -Delectron microscopic examination shows tender capsules and large inclusions of irregular shape inside cells. When grown in media enriched with horse serum, the cells are uniformly stretched and do not differ in polymorphism. When the strain is grown on m-co-peptone agar and protein-depleted media, the cells differ in significant polymorphism: the shape of the cells and their location change. Cells acquire an irregular cocci-shaped, ovoid, dumbbell-shaped, club-shaped, granular form. Cells can be arranged randomly, in clusters, in the form of a paling, rosettes, can be placed around the ring.

Reproduction: transverse division,

splitting up.

Cultural properties. It grows poorly on moc-peptone agar in the form of small (0.5–1.0 mm in diameter) flat colorless colonies of round shape with a dense consistency. The surface is dim, dry, smooth edges smoothed to the surface of the medium. Structure

homogeneous or zernist. Growing into the medium is observed.

It grows well on mono-peptone agar with the addition of 10% horse serum with the formation of isolated or merging colonies of 2-3 mm in diameter, round shape, mucous consistency, shiny.

It grows well on mcr-peptone agar supplemented with 0.2% lysozyme. The growth characteristic is the same as on m-co-peptone agar with the addition of horse serum.

On 3% blood-borne co-peptone agar, growth is scant without hemolysis and no change in the color of the environment.

Potassium tellurium added to the moto-peptone agar in concentrations of 0.01-0.05% does not inhibit the growth of the strain.

Selenic acid sodium added to the medium at a concentration of 0.05% inhibits the growth of the strain.

With growth in m-peptone broth with the addition of 10% horse serum, the strain forms a near-bottom and parietal sediment with simultaneous homogeneous clouding of the medium.

The strain is well preserved on solid nutrient media: mac-peptone agar, mac-peptone agar with 10% horse serum and corresponding liquid nutrient media for 2–3 months at 4 ° C. The strain is well kept in a lyophilized state, without losing its viability, according to available data, for 6 years.

Physiological and biochemical signs. As carbon sources, the strain utilizes: glucose, arabinose, rhamnose, sorbitol, lactose, inositol, xylose, dulcite, mannitol, sucrose. Does not dispose of maltose.

DNA strain contains 35-42% G + C. No cytochrome oxidase system and hem-containing catalase and peroxidase.

Well restores tellurium from. tellurium potassium and does not restore selenium from selenium-acid sodium.

The optimal growth temperature is 3 ° C, the growth temperature range is 20–40 ° C, the growth time is 20–24 hours (depending on the type of nutrient medium). Hydrogen peroxide does not produce. It is not an in vitro antagonist of bacteria.

Attitudes toward antibiotics: resistant to streptomycin - minimal

the inhibitory concentration of the antibiotic (MF) is 61, D4 µg / ml; to lysozyme (IPC-ZOOO micron-g / ml); dioxidine (IPC - 500 µg / ml); dimexide (IPC - 3000 µg / ml). To other drugs sensitive.

Variability. The strain has a high variability of cell morphology when grown on nutrient media depleted in protein.

When the strain population is sown on. nutrient medium (moc-peptone agar); fasting colonies - revertants; clones of which are similar in morphological properties to the original strain

cells on m coagar

horses remain on:

Cocci located in tetrads and clusters of irregular shape, gram-positive Morphologists are the same.

Growth is good, colonies (1-2 mm in diameter) with smooth edges.

puffy

sci

Growth is meager.

Small point

the colonies

Good growth in the form of wall and bottom sediments

Growth good S, large (2-3 mm in diameter) colonies of red color Growth is good, colonies of medium size (1-2 mm in diameter), unpainted Growth is absent

Growth is abundant.

Mycococcus hyperoxydans 167UK, but differ in the predominant expression of oxidase or reductase activity.

Revertant colonies grow on the medium for the 5-10th day. With repeated subcultures, the growth of clones stabilizes during the very first subcultures and is equal to 24-D8 h. I

Table 1 shows a comparative description of the strain Aerococcus vi-ridans (Mycococcus hyperoxydans) GK 308 and the original strain Mycococcus hvperoxydans 16.7 VR

Table 1

Cocci, elongated cocci, ovoid, dumbbell-shaped, club-shaped cells, grains, gram-positive Uniform elongated cocci, ovoid

GROWTH scanty, in the form of napet, dotted flattened colonies with a tendency to grow into the medium

Growth plentiful, drain. Large (2-3 mm in diameter) colonies with even edges. Growth is good in the form of homogeneous opacification, near-wall and bottom sediments. Growth is poor, dotted, colorless colonies.

Medium height, medium sized colonies (Os, 5-1,5 mm in diameter, unpainted)

Growth good Colonies of black color (1-2 mm in diameter) Growth very poor.

Large colonies (2-3 mm in diameter) greening blood agar

40%:

you

ast

n

in

in

1% th

e

Growth growth

Resilient

Resilient

500 500

3.84 2000 250 61.44 3.84 3.84 30.72 30.72 500 500

. 15.36 500 500 1000

30.72 750

Not

5.5 - 6.0 Does not dilute

Does not hydrolyze Does not hydrolyze

+ + + + + + +

Pro; Table 1

Point .colonies. Green environment is absent

Growth growth

Not

Not

0.12 0.12 0.12 61.44 3000 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 0.12 250 1.92 2000

Not

5.8 - 6.1 Does not dilute

Does not hydrolyze Does not hydrolyze

+ + + + + + +

+ + +

+ +

+

Mannitol

Sucrose + G + C content in DNA, strains,% 30-42

The strain GK 308 when grown on myco-peptone agar with the addition of 10% horse serum gives abundant growth, significantly exceeding the growth of the known strain.

Table 2 presents comparative data on the growth ability of strains 167VR and GK 308 with growth on different nutrient media,

Table 2

0.810.1 0.3 ± 0.05

0 0810.01 3.1 ± 0.2

0.0310.006 2.3.10.31

1.3 + 0.1 0.1610.02

This strain is a producer of a wide range of clones with different severity of oxidase and reductase

Reductase

Not expressed 1 LO

Oxidase-reductase

1406156

8 Continuation of table 1

+ +

35 - 42

activity. The different oxidase and reductase activity of the resulting clones is reflected in their properties. Numerous cultures with predominantly oxidase activity are characterized by increased production of hydrogen peroxide, exceeding this characteristic of a known strain (up to, 9.8 mM instead of 2.1 mM, respectively), increased antagonism against pathogenic bacteria.

Pure cultures with predominantly infused reductase activity are characterized by weak production of hydrogen peroxide (up to 0.01 mM) and instead of that strong antagonistic activity against pathogenic bacteria.

The increased variability of strain GK 308 manifests itself in the appearance of different types of colonies when it is sown on a nutrient medium to indicate the oxidase and reductase activity of microorganisms producing hydrogen peroxide.

Table 3 presents data on the frequency of different types of colonies in the natural sieving of the original 167VR and the proposed GK 308 strains.

Table3

Characteristic growth for the bulk of the population 1., 2.10

3.2 -10 -1 -10

1406156

Coloring around

Colorless colonies with black coloring around

Black cols without color zone around

Black cols with black coloration around.

Example 1. Aegos-CUS viridans culture (Mycococcus hyperoxy-dans) GK 308 is grown on mycophone agar with 10% horse serum. A suspension of the daily culture is germinated in a liquid nutrient medium under steady-state conditions at 38 ° C for 10 hours with an initial cell concentration of 1-10 -1-10 per 1 ml of medium.

The germinated suspension is seeded on a nutrient medium to indicate the oxidase and reductase activity of microorganisms producing hydrogen peroxide. Crops are incubated for 6 days at. After incubation, grown colonies with predominantly pronounced oxidase or reductase activity are selected. To obtain clones, colonies are screened on beveled meso-peptone agar and the cultures are incubated for 36 hours at. The resulting clones are examined.

To study the type of predominantly the activity of the clones obtained, streaking of daily cultures (10 cells per 1 ml of physiological sodium chloride solution) was sown on a nutrient medium to indicate oxydase and reductase activity of microorganisms producing hydrogen peroxide. Crops are incubated in a thermostat for 36 hours at 37 ° C, after which they produce results. . Clones with predominantly oxidase activity give an increase in average intensity. Continuation of table 3

1-10 -1-10 1 st

 one

THAT

1-5

sivy with staining in the mobile color of the stroke of growing biomass and the nutrient medium around. The clones with predominantly reductase activity give a good growth with intensive red staining of the biomass stroke. Example 2 Investigate the level of hydrogen peroxide production of the clones obtained. Strain GK 308 hydrogen peroxide does not produce.

Table 4 presents data on the production of hydrogen peroxide, a known strain 167 VK and clones obtained from strain GK 308.

T a b l and c a 4

five

0

167 VR

GK 308

Oxidase

clone

Reductase

clone

Oxidase-reductase

clone

Oxidase 2.1 10.3 Unspecified O

Oxidase 9.8 + 2.3 Reductase 0.1 ± 0.01 Oxidase-0.5 ± 0.02-reductase-1.7 ± 0.4 ny

PRI me R 3. Determine the antagonistic activity of the obtained clones in vitro against pathogenic bacteria of the genera: Proteus, Pseudomonas, Staphylococcus.

eleven

To determine the antagonistic activity of the clones obtained, streak their daily cultures (1 billion cells per 1 ml of physiological sodium chloride solution) onto the surface of meso-peptone agar. Crops are incubated at 37 ° C for 24 hours. In airtight chambers to prevent contamination of the environment, crops are pollinated

The advantages of the strain Aerococcus viridans (Mycococcus hyperoxydans) GK 308 consist in the increased ability to select a wide range of clones with the predominant expression of oxidase or reductase activity, which allows us to consider it

12

suspensions of test bacteria. After 24 hours incubation, growth inhibition zones are considered.

. . ,

Table 5 summarizes the antagonistic activity of the clones obtained in comparison with the known strain 167 VR. Strain GK 308 does not possess in vitro antagonistic activity.

Table3

as a base strain for the selection of biologically active cultures.

Claims (1)

Formula izob eteni The strain of bacteria Aerococcus viridans VNIIA No. 1730 to obtain clones with oxidase and reductase activity,