Hafnia alvei bacteria strain, capable of producing thermolabile lt-enterotoxin

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and can be used to produce thermolabile enterotoxin (LT-enterotoxin) and Hafnia alvei anatoxin when producing a vaccine. The strain is deposited in the State Collection of Pathogenic Microorganisms of FBSI Scientific Centre for Evaluation of Medical Products of the Ministry of Public Health and Social Development of Russia under number 294.

EFFECT: strain has high capacity for producing thermolabile LT-enterotoxin.

1 tbl, 2 ex

 

The present invention relates to biotechnology and can be used for thermolabile of enterotoxin (LT-enterotoxin) and toxoid Hafnia alvei in the production of vaccines.

The technical result of the invention is a strain of Hafnia alvei No. 294, which is isolated from a patient with intestinal infection in 2011 in the Municipal health care institution city Ishimbay of the Republic of Bashkortostan, deposited in the Public collections of pathogenic microorganisms fsbi "Scientific center of expertise of medical application," health Ministry of Russia under No. 294, has a high ability to produce thermolabile LT enterotoxin.

The strains of Hafnia alvei GCPM No. 294 characterized by the following features.

Biochemical properties of the strain belongs to the family Enterobacteriaceae genus Hafnia alvei, because when testing on mikrobiologicheskoi test system version "Vitek 2 systems: 04/01" was consistent with the biochemical properties of this type.

Cultural characteristics.

Grows on conventional dense nutrient medium with the formation of the characteristic of isolated colonies yellowish tint. In mesopatamia the broth is uniform turbidity with precipitation.

The study of virulence factors showed that the strain is rare with complex factors pathoge the activity, can cause acute toxic pneumonia mice (destruction of animals within 3 hours), skin necrosis rabbit (3.5 cm in diameter), swelling of the paws of mice (95 mg), a loop of the small intestine of the rabbit 1,2(V/L), has (alpatyevsky, enterohemolysin), adhesive (positive D-minnesotasvetlana and Dr. minnetrista reaction with chicken erythrocytes), antiinterference, the antilysocyme activities that can protestation test to cause the death of infusoria-shoes within 45 seconds.

This strain is recommended as superproducer thermolabile LT-enterotoxin and as the strain with the complex factors of pathogenicity. The strain can be used in the production of anatoxin and to obtain antitoxic serum.

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 100 mg. Aksenova E.V., 1980

Intranasal introduction of 0.05 ml of a 20-hour broth culture causes acute toxic pneumonia at white mice-males weighing 15-16 g (death within 4 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 3 see

In the experience of a loop of the small intestine of the rabbit with the introduction of 0 million M.L. 20-hour broth culture gives a positive result (V/L=l,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).

Storage conditions of culture after freeze drying in vials or grown at 37°C 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°C for 24 hours.

Genetic characteristics: HLy+, Ent+, MS+, MR+, LT+ALA+, AKA+. Resistant to ampicillin, ampicillin/sulbactam, cefuroxime sensitive to fluoroquinolones.

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), incubated in a thermostat at 37°C for 20 hours, centrifuged at 6000 rpm for 7 minutes. The obtained supernatant (centrifugal) in a volume of 0.05 ml is injected subplantar in his left rear leg of male mice weighing 15-18 g After 24 hours the animals killed by ether and missing limbs on the ankle pads are weighed on a torsion balance, with centrifugal strain the Hafnia alvei No. 294 gave the difference between the left and right legs at least 100 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°C centrifugal 20-hour broth culture of 30-35 mg, respectively.

The results are shown in the table.

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. 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 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 strain Hafnia alvei No. 294 was 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 100°C 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
Comparative figures LT-antireproductive activity Hafnia alvei
The number of strain Swelling of the legs (mg)Isolated loop of the small intestine (V/L)
6Hafnia alvei No. 2941001,3
8--740,9
7--760,9
3--801,0
9--851,0
1--750,8
3--700,7
4--780,9
Control30of 0.25-0.3
Note: the table shows the averages and the five edema.

The strain of bacteria Hafnia alvei, with the ability to produce thermolabile LT enterotoxin deposited in the Public collections of pathogenic microorganisms III-IV groups of pathogenicity fsbi "Scientific center of expertise of medical application," health Ministry of Russia under No. 294.



 

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