Bacterial strain enterobacter agglomerans risk n 257, with complex pathogenicity factors

 

(57) Abstract:

The invention is intended to obtain thermolabile of enterotoxin (LT enterotoxin) and toxoid Enterobacter agglomerans in the production of vaccines. The strain of Enterobacter agglomerans N 5356 isolated from the stool of a patient with mycosis of the feet, 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 257. The strain of Enterobacter agglomerans risk N 257 characterized by the following features. When grown on nutrient media at 37°C, the strain on the surface of the medium increases with the education characteristic of isolated colonies with a yellowish tinge. When grown in broth of Hottinger (pH of 7.2) at 37°C for 24 h causes a uniform turbidity with precipitation. Subplantar administration to mice weighing 15-16 g 0.05 ml of centrifugate 20-hour broth culture causes swelling of the legs more than 95 mg of Intranasal introduction of 0.05 mg 20-hour broth culture causes the death of 95% of male mice weighing 15-16 g for 4 h at phenomena tonic convulsions and asphyxia. Intradermal injection of 300 million microbial cells 20-hour broth culture calls n the term culture gives a positive result (V/L=1,2). Heated at 56°C for 45 min centrifugal 20-hour broth culture causes swelling of the paws of mice up to 30 mg without causing necrosis of the skin of the rabbit. Heated at 100°C 20-hour culture does not cause accumulation of fluid in the isolated loop of the small intestine of the rabbit (V/L=0,3). Medium for reproduction - broth of Hottinger (pH of 7.2) containing 5% rabbit erythrocytes at 37°C for 24 h Genetic features: ly+, Ent+ MS+ MR+. Resistant to penicillin, tetracycline, erythromycin, chloramphenicol, carbenicillin, rifampin, oleandomycin. The strain of Enterobacter agglomerans risk N 257 has a high toxin-producing ability. table 1.

The invention relates to biotechnology and can be used for thermolabile of enterotoxin (LT enterotoxin) and toxoid Enterobacter agglomerans, in the manufacture of vaccines.

The technical result is a strain of Enterobacter agglomerans N 5356, with increased toxinproducing ability.

The strain of Enterobacter agglomerans N 5356 isolated from the stool of a patient with mycosis stop in 1998 in the city hospital, Ishimbay of the Republic of Bashkortostan, deposited in the collection of the State research Institute of standardization and control of medical N 257 characterized by the following features:

Cultural characteristics.

When grown on nutrient media at 37oC strain on the surface of the medium increases with the education characteristic of isolated colonies with a yellowish tinge. When grown in broth of Hottinger (pH of 7.2) at 37oC for 24 h causes a uniform turbidity with precipitation.

Methods for determining the activity of LT-enterotoxin:

Subplantar administration to mice weighing 15-16 g 0.05 ml of centrifugate 20-hour broth culture causes swelling of the legs more than 95 mg (Aksenova E. C. 1980).

Intranasal introduction of 0.05 mg 20-hour broth culture causes the death of 95% of male mice weighing 15-16 g for 4 h at phenomena tonic convulsions and asphyxia (Voino-Yasenetsky N. To. 1967).

Intradermal injection of 300 million microbial cells 20-hour broth culture causes necrosis of the skin of a rabbit with a diameter of 3 see

In the experience of a loop of the small intestine of the rabbit with the introduction of 500 million MT 20-hour broth culture gives a positive result (V/L =1,2).

Heated at 56oC for 45 min, centrifuged 20-hour broth culture causes swelling of the paws of mice up to 30 mg without causing necrosis of the skin of the rabbit. Heated at 100oC 2SS="ptx2">

Storage conditions of culture after leofiles drying when grown in 0.3% MPA (pH 7,2), filled with sterile vaseline oil, at a temperature of 4oC.

Medium for reproduction - broth of Hottinger (pH of 7.2) containing 5% rabbit erythrocytes at 37oC for 24 h

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

Resistant to penicillin, tetracycline, erythromycin, chloramphenicol, carbenicillin, rifampin, oleandomycin.

Example 1. 0.1 ml of 1 billion suspension daily agar culture in physiological solution sown in sterile broth of Hottinger (5 ml, pH of 7.2) and incubated in a thermostat at 30oC for 20 hours, centrifuged at 6000 rpm for 7 min Obtained supernatant (centrifugal) in a volume of 0.05 ml is injected subplantar in his left rear leg of male mice weighing 15-16, After 24 h the animals killed by ether and missing limbs on the ankle pads are weighed on a torsion balance, with centrifugal strain Enterobacter agglomerans risk N 257 gave the difference between the left and right legs over 95 mg, the remaining 20 strains capable of producing LT enterotoxin from 65 to 79 mg Controls - native sterile broth of Hottinger (pH 7,2) and heated at 56oC C the LASS="ptx2">

Example 2.

0.1 mg 1 billion suspension daily agar culture sow in the broth of Hottinger (pH of 7.2), incubated in a thermostat at 30oC for 20 h and 500 million MT suspension in a volume of 1 ml is injected into the isolated segment of the loop of the small intestine of the rabbit. After 16 h of animals killed by air embolism and calculate the ratio of the volume of exudate to the length of the segment (V/L). This 20-hour broth culture of the strain of Enterobacter agglomerans risk N 257 were able to give an accumulation of fluid in large quantity and rate V/L amounted to 1.2. The remaining 20 strains capable of producing LT enterotoxin, metric V/L ranged from 0.9 to 1.0. With the introduction of sterile broth of Hottinger and Greta at 100oC 20-hour broth culture of the ratio of the volume of accumulated fluid to the segment length was 0.25 and 0.3, respectively.

Bacterial strain Enterobacter agglomerates risk N257 with complex factors of pathogenicity.

 

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FIELD: biotechnology, microbiology, medicine.

SUBSTANCE: invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea.

EFFECT: valuable medicinal properties of strain.

5 cl, 8 dwg, 10 ex

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