RU2113471C1 - Strain of bacterium proteus mirabilis - a producer of thermolabile enterotoxin - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: strain Proteus mirabilis N 2536 is isolated from patient with urological infection in 1992 and deposited in Collection of The State Scientific-Research Institute of standardization and control of medicinal biological preparation at number 237. Genetic properties of the strain: strain shows resistance to penicillin, neomycin, monomycin, polymyxin, tetracycline, lyncomycin, erythromycin, ristomycin, oxacilline, levomycetin. Strain shows increased toxin-producing capability. Strain can be used for preparing thermolabile enterotoxin and anatoxin from Proteus mirabilis at vaccines production. EFFECT: new strain indicated above, increased production of toxins. 1 tbl

Description

 The invention relates to biotechnology and can be used to obtain thermolabile enterotoxin / LT-enterotoxin / and toxoid Proteus mirabilis in the production of vaccines.

 The aim of the invention is a strain of Proteus mirabilis 2536, with increased toxin-producing ability.

 Strain P. mirabilis N 2536 was isolated from a patient with a urological infection in 1992 and was deposited in the collection of the State Research Institute for Standardization and Control of Medical Biological Preparations named after L.A. Tarasevich and has a registration number 237.

The strain Proteus mirabilis GISK N 237 is characterized by the following features:
Morphological signs - when stained according to Gram, gram-negative sticks are located in a smear separately or in pairs. In the native drug are motile.

Cultural signs: when grown on 1.5% meat-pentone agar / pH 7.2-7.4 / containing 50 rabbit erythrocytes, at 35-36 ^o C the strain on the surface of the nutrient medium gives a characteristic creeping growth in the form of a veil with a zone hemolysis. When grown in Hottinger broth (pH 7.2-7.4 /, temperature 35-36 ^o C, for 20 h gives a diffuse opacification and a delicate film on the surface of the medium.

 It forms indole phenylalanine dezenose, does not ferment lactose and ferments with the formation of acid and gas glucose, xylose, salicin, maltose, sucrose, splits urea, does not produce oritin decarboxylase.

 Serological properties.

 A 20-hour agar culture yields agglutination with only HA 4 polyvalent diagnostic protein rabbit dry whey.

Methods for determining the activity of LT enterotoxin:
- subplanar administration to mice weighing 15-16 g of 0.05 centrifugate of a 20-40-hour broth culture causes paw edema of at least 110 mg (Aksenova E.V., 1980);
- intranasal administration of 0.05 mg of a 20-40 hour broth culture causes the death of 95% of male mice weighing 15-16 g for 20 minutes with the phenomena of tonic seizures and asphyxia (Voino-Yasenetskaya N.K., 1957);
- intramuscular injection of 300 million microbial cells to a 20-hour broth culture causes necrosis of rabbit skin with a diameter of more than 3.5 cm;
- in the experiment, the loop of the small intestine of the rabbit with the introduction of 500 million MT 20-hour broth culture gives a positive result;
- a centrifugate heated at 56 ^° C for 45 minutes, a 20-hour broth culture causes the paw edema of mice up to 30 mg, does not cause rabbit skin nicrosis. Heated at 100 ^o C 20-hour broth culture does not cause accumulation of fluid in an isolated loop of the small intestine of the rabbit / v / l 0,3 /.

Storage conditions of the culture after leophilic drying when grown in 0.3% MPA / pH 7.2-7.4 / filled with sterile liquid paraffin, at 4 ^o C.

Propagation media - Hottenger broth / pH 7.2-7.4 / containing 5% rabbit erythrocytes at a temperature of 35-36 ^o C for 20 hours

 Genetic features: resistant to penicillin, neomycin, monomycin, polymyxin, tetracycline, lincomycin, erythromycin, ristomycin, oxacillin, chloramphenicol.

Example 1. 0.1 ml of 1 billion suspension of daily agar culture in physiological solution is seeded in sterile Hottinger broth / 5 ml of pH 7.2-7.4 / incubated in an incubator at 36 ^o C for 20 h, centrifuged at 6000 r / min for 7 minutes The resulting supernatant / centrifugal / in a volume of 0.05 ml was introduced subplanarly into the hind left paw of male mice weighing 15-16 g. After 24 hours, the animals were sacrificed with ether and the amputated amyloid paws were weighed on a torsion balance. In this case, the centrifugate of the Proteus mirabilis strain GISK N 237 gave a difference between the left and right paws of at least 110 ml, the remaining 20 strains capable of producing RT enterotoxin from 65 to 90 mg. Hottinger control sterile broth / PH 7.2 / 7.4 / and a centrifugate of 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 daily agar culture is sown in Hottinger broth / pH 7.2-7.4 / incubated in an incubator at 36 ^o C for 20 hours and 500 million mt suspension in a volume of 1 ml injected into a segment of an isolated loop of a rabbit's small intestine. After 16 hours, the animals are sacrificed by air embolism and the ratio of the volume of exudate to the length of the segment / v / l / is calculated. At the same time, the 20-hour broth culture of the Proteus mirabilis strain GISK 237 was able to accumulate large amounts of liquid and the v / l index was 1, 2. In the remaining 20 strains capable of producing LT enterotoxin, the v / l index ranged from 0 9 - 1.0. With the introduction of sterile Hottinger broth and heated at 100 ^o C for 20 hours of broth culture, the ratio of the accumulated liquid volume and the segment length was 0.25 and 0.3, respectively.

Claims (1)

 The bacterial strain Proteus mirabilis GISK 237 is a producer of thermolabile enterotoxin.

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