The strain of bacteria citrobacter freundii risk no. 255 with complex pathogenicity factors

 

(57) Abstract:

The invention relates to biotechnology and can be used for thermolabile of enterotoxin and toxoid Citrobacter freundii in the production of vaccines. The strain of bacteria Citrobacter freundii has a complex of factors of pathogenicity, as well as increased toxinproducing ability.

The invention relates to biotechnology and can be used for thermolabile of enterotoxin (LT-enterotoxin) and toxoid in the vaccine production, and can also be used as a reference strain of Citrobacter freundii.

The technical result is a strain of Citrobacter freundii 882 N with complex pathogenicity factors.

The strain was isolated from the faeces of a patient with acute leukemia in 1998 in the city clinical hospital # 8, Ufa and deposited in the collection of the state research Institute of standardization and control of medical biological preparations. L. A. Tarasevich and has a registration number 255.

Citrobacter freundii risk N 255 characterized by the following features.

Cultural characteristics: grows on conventional dense nutrient medium with obray uniform turbidity with precipitation of uniform sediment.

Methods for determining the activity of LT-enterotoxin:

Subplantar (model edema feet) administration to mice weighing 15-16 g 0.05 ml of centrifugate 20-hour broth culture causes swelling of the legs with a difference of more than 120 mg.

Intranasal (Voino-Yasenetsky N. To. 1967) introduction 80 million MT cause the death of 95% of white mice-males weighing 15-16 g for 20 minutes.

Intramuscular injection of 300 million microbial cells 20-hour broth culture causes necrosis of the skin of a rabbit with a diameter of 3.2, see

In the experience of the loops of the small intestine of the rabbit with the introduction of strain at a concentration of 500 million MT gives a positive result (V/L = 1,4).

Heated at 56oC for 45 min centrifugal 20-hour broth culture causes swelling of the paws of mice up to 30 mg does not cause necrosis of the skin of the rabbit. Heated at 100oC 20-hour culture does not cause accumulation of fluid in the isolated loop of the small intestine of the rabbit (V/L = 0,3).

Storage conditions: at a temperature of 4oC culture after leofiles drying or grown in 0.3% MPA (pH 7,2-7,4) and filled in sterile vaseline oil. Conditions and composition of the medium for reproduction - broth of Hottinger (pH 7,2-7,4), 1.5% of MPA at a temperature of 37oC for 18-24 hours.

An example is nger (5 ml pH 7,2-7,4), incubated in an incubator at 36oC for 20 hours, centrifuged at 6000 rpm for 7 minutes. The obtained supernatant (centrifuged in a volume of 0.05 ml is injected subplantar in his left rear leg of male mice weighing 15-16, After 24 hours the animals killed by ether and missing limbs on the ankle pads are weighed on a torsion balance. However centrifugal strain GISC N 255 gave the difference between the left and right legs not less than 120 mg. Controls - native sterile broth of Hottinger (pH 7,2-7,4) and heated at 56oC centrifugal 20-hour broth culture of 30-35 mg, respectively.

Example 2. Daily agar culture was diluted in physiological solution, while creating different concentrations of microbial cells in 1 ml: 1 billion MT , 800 million MT, 600 million MT ... up to 25 million MT of male Mice weighing 16-18 g intraperitoneally infected with different concentrations of microbial bodies, to determine LD50. LD50from Citrobacter freundii risk N 255 amounted to 50 million MT

The strain of bacteria Citrobacter freundii GIS 255 with complex factors of pathogenicity.

 

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