Microorganism strain klebsiella pneumoniae gisk №278 as producer of lysozyme inhibitor

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to the microorganism strain Klebsiella pneumoniae GISK № 278 isolated from a patent feces suffering from intestine dysbacteriosis. The strain is used for preparing an agent for producing the lysozyme inhibitor. The level of activity of lysozyme inhibitor produced by this strain is 1.64-2.16 mcg/ml of *OD value.

EFFECT: valuable properties of strain.

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The invention relates to Microbiology and can be used to obtain an inhibitor of lysozyme.

Analogs of the invention in the patent and scientific literature is not found.

The technical result, which is aimed invention is to obtain funds for the production of an inhibitor of lysozyme.

This technical result is achieved by a new strain of Klebsiella pneumoniae GISC No. 278.

A strain of Klebsiella pneumoniae GISC No. 278 isolated from the stool of a patient with intestinal dysbacteriosis in a survey of microbiocenosis of the distal colon in 2004 in the laboratory of biomonitoring and molecular genetic studies of ICS Orenburg, Ural branch of RAS.

The resulting strain of Klebsiella pneumoniae GISC No. 278 deposited in the collection of the State research Institute of standardization and control of medical biological preparations. Laurasia and has a registration number 278.

A strain of Klebsiella pneumoniae GISC No. 278 characterized by the following cultural-morphological and physiological-biochemical characteristics.

Cultural morphological traits:

Cells are gram-negative, still sticks forming the capsule size (0,3...1,0)×(6,0 0,6...) microns.

Mastopathy agar (37°, 48 hours). Form isolated wet, slit is simple colony of white or gray-white in color. Culture "stretches" the capture from the surface of the agar microbiological loop.

Meat-peptone broth (37°, 48 hours). Without shaking causes uniform turbidity and sedimentation when grown for 18-24 hours. With shaking on a rocking chair medium becomes uniform turbidity without sediment.

Physiological and biochemical characteristics:

Optional gone anaerobic.

Temperature range for growth 25-42°C. the Optimum temperature for growth 28-39°C. the Maximum growth rate at 37-39°C.

The optimum pH 7,2-7,6.

The sulfide - negative, mannitol - positive, lysine - positive, indole negative, ornithine - negative, urease - positive, arginine - negative, citrate Simons - positive, malonate sodium - positive, phenylalanine - negative, β-galactosidase - positive, Inositol - positive, adonit - positive, cellobiose - positive, sucrose - positive, trehalose - positive, mannitol - positive, acetoin - positive esculin - positive, sorbitol - positive, rhamnose - positive, raffinose - positive, dulcet - positive, glucose positive reaction Voges-Proskauer - positive.

Storage conditions at a temperature of 4°culture after freeze drying or grown in 0.3% MPA (pH 7,2-7,6) Italica sterile vaseline oil.

Conditions and the composition of the medium for the propagation of BCH - T 32-33°With (pH 7,2-7,6), 1,5% MPA at T 37°C for 18-24 hours.

A strain of Klebsiella pneumoniae GISC No. 278 identified in the "Determinant bacteria Burgi" (1997) as the type strain of Klebsiella pneumoniae.

The level of activity produced by Klebsiella pneumoniae GISC No. 278 inhibitor of lysozyme is determined according to a known method for determining the antilysocyme activity (patent RF №2126051, published. 23.10.1990).

The essence of the invention is illustrated by the following specific examples of strains of Klebsiella pneumoniae GISC No. 278.

Example 1

Broth culture of a strain of Klebsiella pneumoniae GISC No. 278 was obtained by cultivation of this strain in test tubes in a liquid nutrient medium of the following composition: peptone - 10.0 g; NaCl 5.0 g; water meat - 1000,0 ml, pH 7.2 to 7.4. Temperature 37°C, the incubation period of 18-24 hours. The obtained broth culture was centrifuged at 3000 rpm for 20 minutes. The culture fluid was separated from the bacterial cells sterile glass pipette into a separate container. This culture liquid is a crude drug inhibitor of lysozyme. The presence and level of activity produced by a strain of Klebsiella pneumoniae GISC No. 278 inhibitor of lysozyme was determined as follows: 0.9 ml of the obtained culture liquid was mixed with 0.1 ml of a solution of lysozyme and incu is Aravali 60-120 minutes at 37° C. Then 0.5 ml of a mixture of the culture fluid and lysozyme was added to 2.0 ml of a suspension of the test culture of Micrococcus luteus ATCC 15307 and measured the optical density of the mixture after 30 and 150 with the spectrophotometer. As control was used a mixture of nutrient broth and lysozyme.

On the availability produced by a strain of Klebsiella pneumoniae GISC No. 278 inhibitor of lysozyme was judged by lysis of the culture test Micrococcus luteus ATCC 15307. The level of activity produced by a strain of Klebsiella pneumoniae GISC No. 278 inhibitor of lysozyme was calculated by the formula: A=V1*C1(1-D0/Dk)/V2*Y, where a is antilysocyme activity derived inhibitor of lysozyme (μg/ml*OP), V1- volume of a solution of lysozyme initial concentration, V2- the volume of supernatant broth culture. With1- initial concentration of the lysozyme, Y is the optical density of the broth culture, D0the change in optical density of the test culture of Micrococcus luteus was ATSS 15307 between 30 and 150 seconds in the experiment, Dkthe change in optical density of the test culture of Micrococcus luteus ATCC 15307 between 30 and 150 seconds in the control.

The level of activity of an inhibitor of lysozyme produced by a strain of Klebsiella pneumoniae GISC No. 278 ranged from A=(1-0,032/0,125)*2,2=1,64 up to A=(1-0,0025/0,125)*2,2=2,16.

Example 2

Analogously to example 1 was performed experiments to determine the presence and level of activity inhibitor of lysozyme produced by the strain is m Klebsiella pneumoniae GISC No. 278 and wild strains of Klebsiellae sp., isolated from the intestinal habitat.

In the study of the level of activity of an inhibitor of lysozyme produced by a strain of Klebsiella pneumoniae GISC No. 278 and wild strains of Klebsiellae sp., isolated from the intestinal habitat, the authors experimentally found that the level of activity of an inhibitor of lysozyme produced by a strain of Klebsiella pneumoniae GISC No. 278 higher compared to the level of activity of an inhibitor of lysozyme produced by wild strains of Klebsiellae sp., which reflects the higher productivity of the proposed strain of Klebsiella pneumoniae GISC No. 278 compared with wild strains of Klebsiellae sp.

The research results are summarized in the table.

Table

The comparative characteristic of the level of activity of an inhibitor of lysozyme produced by a strain of Klebsiella pneumoniae GISC No. 278 and wild strains of Klebsiellae sp.
The analyzed strains of KlebsiellaThe level of activity of an inhibitor of lysozyme (μg/ml*OP)
0,20,40,60,81,01,21,41,61,82,02,2
Klebsiella D1+------ ---
Klebsiella D2++++++-----
Klebsiella D3++---------
Klebsiella D4++++------
Klebsiella D5+----------
Klebsiella D6+++-------
Klebsiella d++++-------
Klebsiella d+++ ++-----
Klebsiella D9++---------
Klebsiella D10

Klebsiella D11
++++-------
+----------
Klebsiella risk No. 278+++++++++++
Klebsiella D1...D11 - wild strains Klebsiellae sp.

Bacterial strain Klebsiella pneumoniae GISC No. 278 - producer inhibitor of lysozyme.



 

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