RU2249038C1 - Strain of bacterium serratia marcescens gisk 268 as producer of thermolabile enterotoxin - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: the strain of microorganism Serratia marcescens GISK N 268 - a producer of thermolabile enterotoxin is isolated from a patient with wound infection. Isolation of the new strain provides expanding assortment of strains as producers of thermolabile enterotoxin. The strain shows high productivity.

EFFECT: valuable properties of strain.

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Description

The invention relates to biotechnology and can be used to obtain thermolabile enterotoxin (RT-enterotoxin) and toxoid Serratia marcescens in the production of vaccines.

The technical result is a strain of Serratia marcescens No. 892 - isolated from a patient with wound infection in the Republican Clinical Hospital named after G.G. Kuvatova, Ufa, deposited in the collection of the State Research Institute of Standardization and Control of Medical Biological Preparations named after L.A. Tarasevich and has a registration number 268.

Strain Serratia marcescens GISK No. 268 is characterized by the following features:

Cultural signs

When grown on ordinary nutrient media, at a temperature of 37 ° C, the strain on the surface of a dense nutrient medium grows with the formation of characteristic isolated colonies and red-raspberry pigment during the day. When grown in Hottinger broth (pH 7.2-7.4), at a temperature of 37 ° C for 24 hours, it causes uniform clouding with red-crimson light and precipitation.

Methods for determining the activity of LT enterotoxin:

Subplant implantation in mice weighing 15-16 g of 0.05 ml centrifugate of a 20-hour broth culture causes paw edema of more than 100 mg. Aksenova E.V., 1980

Intranasal administration of 0.05 ml of a 20-hour broth culture causes acute toxic pneumonia in white male mice weighing 15-16 g (death within 4 hours). Voyno-Yasenetskaya N.K., 1957

Intramuscular administration of 300 million microbial cells of a 20-hour broth culture causes necrosis of rabbit skin 1.5 cm in diameter.

In the experiment, the loop of the small intestine of the rabbit with the introduction of 80 million MT A 20-hour broth culture gives a positive result (V / L = 1.2).

Heated at 56 ° C for 45 min, a centrifugate of a 20-hour broth culture causes swelling of the paws of mice up to 30 mg without causing necrosis of rabbit skin. A 20-hour culture heated at 100 ° C does not cause fluid accumulation in an isolated loop of the rabbit small intestine (V / L = 0.3).

Storage conditions of the culture after freeze drying in an ampoule or grown at 37 ° C on 0.3% MPA (pH 7.2-7.4), filled with sterile liquid paraffin, at a temperature of 4 ° C.

Propagation media - Hottinger broth (pH 7.2), meat-peptone agar containing 5% rabbit erythrocytes at a temperature of 37 ° C for 24 hours.

Genetic features: Hly +, Ent +, MS +, MR +.

Resistant to penicillin, oleandomycin, rifampicin, fromilid, erythromycin, furagin.

Example 1

0.1 ml of a 1 billion suspension of daily agar culture in physiological solution is sown in a sterile Hotinger broth (5 ml pH-7.2), incubated in an incubator at 37 ° C for 20 hours, centrifuged at 6000 rpm for 7 minutes . The resulting supernatant (centrifugate) in a volume of 0.05 ml is introduced subplanarly into the hind left foot of male mice weighing 15-16 g. After 24 hours, the animals are sacrificed with ether and the amputated amyloid paws are weighed on a torsion balance, while the centrifugate of the Serratia marcescens strain GISK No. 268 yielded a difference between the left and right paws of at least 100 mg, the remaining 20 strains capable of producing LT enterotoxin ranged from 65 to 90 mg. Controls - a native sterile Hottinger broth (pH 7.2) and a centrifugate of a 20-hour broth culture 30-35 mg heated at 56 ° C, respectively.

The results are shown in the table.

Example 2

0.1 ml of 1 billion suspension of the daily agar culture was sown in Hottinger broth (pH 7.2), incubated in an incubator at 37 ° C for 20 hours and 80 million M.T. a suspension in a volume of 1 ml is introduced into the segment of an isolated loop of the rabbit small intestine. After 16 hours, the animals are sacrificed by air embolism and the ratio of exudate volume to segment length (V / L) is calculated. At the same time, the 20-hour broth culture of the strain Serratia marcescens GISK No. 268 was able to accumulate a large amount of liquid and the V / L index was 1.2. In the remaining 20 strains, the LT / enterotoxin capable of producing V / L ranged from 0.9-1.0. With the introduction of sterile Hottinger broth and 20-hour broth culture heated at 100 ° С, the ratio of the accumulated liquid volume to the segment length was 0.25 and 0.3, respectively.

Comparative indices of the RT enteroproducing activity of Serratia

Strain number View Paw edema (mg) Isolated Small Intestine Loop (V / L) 892 GISK No. 268 100 1,2 101 S.marcescens 74 0.8 324 - \ - 76 0.9 44 - \ - 80 1,0 72 - \ - 85 1.0 1065 S.odorifera 75 0.8 5 - \ - 70 0.7 thirteen - \ - 78 0.9 The control thirty 0.25-0.3

Note: the table shows the average data from five edema.

Claims (1)

The bacterial strain Serratia marcescens GISK No. 268 is a producer of thermolabile enterotoxin.