Strain of microorganism serratia odorifera gisk 275 as producer of thermolabile enterotoxin

FIELD: biotechnology, microbiology.

SUBSTANCE: strain is isolated from patient with urological infection. The strain elicits high productivity and can be used in preparing vaccines.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

 

The invention relates to biotechnology and can be used for thermolabile of enterotoxin (LT enterotoxin) and toxoid Serratia odorifera in the production of vaccines.

The technical result is a strain of Serratia odorifera No. 519, isolated from a patient with wound infection in the Republican clinical hospital n.a. How Ufa, deposited in the collection of the State research Institute of standardization and control of medical biological preparations. Laurasia and has a registration number 275.

Strain Serratia odorifera risk No. 275 characterized by the following features:

Cultural characteristics.

When grown on nutrient media at a temperature of 37°With the strain on the surface of a dense nutrient medium increases with the education characteristic of isolated colonies and red-crimson pigment during the day. When grown in broth of Hottinger (pH 7,2-7,4), at a temperature of 37°within 24 hours causes a uniform haze with red-crimson light and 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 105 mg Aksenova E.V. 1980

Intranasal introduction of 0.05 ml of a 20-hour broth culture causes acute the Yu toxic pneumonia at white mice-males weighing 15-16 g (death within 5 hours). Voyno-Yasenetsky NICHOLAS 1957

Intramuscular injection of 300 million microbial cells 20-hour broth culture causes necrosis of the skin of a rabbit with a diameter of 1.6, see

In the experience of a loop of the small intestine of the rabbit with the introduction of 80 million M.L. 20-hour broth 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°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 of culture after freeze drying in vials or grown at 37°With 0.3%MPA (pH 7,2-7,4), filled with sterile vaseline oil, at a temperature of 4°C.

Medium for reproduction - broth of Hottinger (pH 7,2), mesopatania agar containing 5% rabbit erythrocytes at 37 ° °With within 24 hours.

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

Resistant to penicillin, oleandomycin, fromilid, erythromycin, furagin, tetracycline, polymyxin.

Example 1.

0.1 ml of 1 billion suspension daily agar culture in physiological solution sown in sterile broth, Hottinger (5 ml, pH of 7.2) and incubated in a thermostat at 37°C for 20 hours, centrifuged at 6000 rpm for 7 minutes. The obtained supernatant (centrif the gat) 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 Galena-ankle joint pads are weighed on a torsion balance, with centrifugal strain Serratia odorifera risk No. 275 gave the difference between the left and right legs not less than 105 mg, the remaining 20 strains capable of producing LT enterotoxin from 65 to 90 mg. Controls - native sterile broth of Hottinger (pH 7,2) and heated at 56°centrifugal 20-hour broth culture of 30-35 mg, respectively.

The results are shown in table 1.

Example 2.

0.1 ml of 1 billion suspension daily agar culture sow in the broth of Hottinger (pH of 7.2), incubated in a thermostat at 37°C for 20 h and 80 million M.L. suspensio in a volume of 1 ml is injected into the isolated segment of the loop of the small intestine of the rabbit. After 16 hours the 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 Serratia odorifera risk No. 275 was able to give an accumulation of fluid in large quantity and rate V/L were 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 100°20-hour broth culture of the ratio of the volume of accumulated fluid to the segment length was 0.25 and 0.3, respectively.

Table No. 1.

Comparative figures LT-antireproductive activity of Serratia.
The number of strainSwelling of the legs (mg)Isolated loop of the small intestine (V/L)
519Risk No. 2751051,2
101S.marcescens740,8
324--760,9
44--801,0
72--851,0
1065S.odorifera750,8
5--700,7
13--780,9
Control 30of 0.25-0.3
Note: the table shows the averages of the five edema.

The bacterial strain Serratia odorifera risk No. 275 - producer of thermolabile enterotoxin.



 

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