Hafnia alvei bacteria strain, capable of producing thermolabile lt-enterotoxin. RU patent 2514656.

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.

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

The technical result of the invention is a strain of Hafnia alvei №294, isolated from a patient with an intestinal infection in 2011 at the Municipal institution of public health of the city of Ishimbay of the Republic of Bashkortostan, deposited in the State collection of pathogenic microorganisms, St Petersburg Scientific centre of medical products Ministry of health of Russia under number 294, has a high capacity to produce thermolabile LT-enterotoxin.

Strain Hafnia alvei GKM №294 characterized by the following features.

On biochemical properties strain belongs to the family Enterobacteriaceae kind of Hafnia alvei, because when tested on mikrobiologicheskoe test-system version "Vitek 2 Sistems: 04/01" consistent with biochemical properties of this species.

Cultural characteristics.

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

The study of virulence factors showed that the strain is rare with complex the pathological factors that can cause acute toxic pneumonia mice (the death of animals within 3 hours), necrosis of the skin rabbit (3.5 cm in diameter), swelling of the legs of mice (95 mg), a loop of small bowel rabbit 1,2(V/L), has (alpatovsky, enterohemolysin), adhesive (a 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 the recommended superproducer thermolabile LT-enterotoxin and as a strain with complex pathogenicity factors. The strain can be used in the production of anatoxinum and to obtain antitoxic serum.

The methods for determining the activity LT-enterotoksina.

Subplantar introduction to mice weighing 15-16 g 0.05 ml of centrifugate 20-hour broth culture causes swelling legs over 100 mg. Aksenova E.V., 1980,

Intranasal introduction of 0.05 ml of 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 300 million microbial cells 20-hour broth culture causes necrosis of the skin rabbit diameter 3 see

In the experience of the loop of small bowel rabbit with the introduction of 80 million M.L. 20-hour broth culture gives a positive result (V/L=L,2).

Thermally treated at 56 degrees C for 45 min of centrifugal 20-hour broth culture causes swelling of the legs of mice up to 30 mg without causing necrosis skin rabbit. Thermally treated at 100 C 20-hour culture is not causing the accumulation of fluid in an isolated loop of small bowel rabbit (V/L=0,3).

Storage conditions of culture after the freeze drying vials or grown at 37 C by 0.3%MPA (pH of 7.2 to 7.4), filled with sterile vaseline oil at a temperature of 4o C.

Medium for reproduction - broth of Hottinger (pH 7,2), massoperenos agar containing 5% of erythrocytes rabbit at a temperature of 37 C for 24 hours.

Genetic features: HLy+, Ent+MS+MR+LT+ALA+, AKA+. Resistant to ampicillin, ampicillin/sulbactam, aporoximately sensitive fluoroquinolone.

Example 1.

0.1 ml of 1 billion suspension daily agar culture in physiological solution sow in sterile broth, Hottinger (5 ml pH 7,2), incubated in thermostat at 37 C for 20 hours, centrifuged at 6000 rpm within 7 minutes. Received the supernatant (centrifugal) in the amount of 0.05 ml injected subplantar in his left rear leg of male mice weighing 15-18, 24 hours animals kill ether and missing limbs on the ankle foot weighed on torsion scales, with centrifugal strain Hafnia alvei №294 given the difference between the left and right legs not less than 100 mg and 20 strains that can produce LT-enterotoxin from 65 to 90 mg Controls - native sterile broth of Hottinger (pH 7,2) and warmed up at 56 degrees From centrifugal 20-hour broth culture 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 7,2), incubated in thermostat at 37 C for 20 hours, and 80 million M.L. suspension in the volume of 1 ml is injected in an isolated segment of the loop of small bowel rabbit. After 16 hours of animals killed by an air embolism and calculate the ratio of the exudate to the length of the segment (V/L). This 20-hour broth culture of strain Hafnia alvei №294 were able to give an accumulation of fluid in a large number and indicator V/L was 1.2. The remaining 20 strains that can produce LT-enterotoxin figure 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, the volume of accumulated fluid to the length of the segment was 0.25 and 0.3 respectively.

Comparative indicators LT-interpretiruya activity Hafnia alvei

Room strain

Swelling of feet (mg)

Isolated loop of the small intestine (V/L)

Hafnia alvei №294

0,25-0,3

Note: the table shows the average data of five swellings.

The strain of bacteria Hafnia alvei, with the capacity to produce thermolabile LT-enterotoxin deposited in The state collection of pathogenic microorganisms III-IV groups of pathogenicity, St Petersburg Scientific centre of medical products Ministry of health of Russia under number 294.