Strain regionalnego bacteriophage scientifically active against legionella pneumophila, l. micdadei, l. dumoffii, and l. bozemanii

 

The invention relates to medical Microbiology and can be used to identify the causative agents of Legionnaires allocated from the body of the patient and infected objects of the external environment. Strain regionalnego bacteriophage can be used to identify the main types of pathogens Legionnaires ' disease Legionella pneumophila, L. micdadei, L. dumoffii, and L. bozemanii laboratory diagnosis of legionellosis isolated from organs of laboratory animals infected with typical strain Philadelphia-1 L. Pneumophila, and deposited in the collection of phage research Institute of Microbiology of the Ministry of defense under the designation scientifically. The use of bacteriophage allows to increase the specificity of the identification of the causative agent of legionellosis from other pathogens. 3 Il.

The invention relates to medical Microbiology, namely by means of laboratory diagnosis of legionellosis, and can be used in scientific and practical laboratories identification of the causative agents of Legionnaires allocated from the body of the patient and infected objects of the external environment.

There are many known bacteriophages used to identify and faguibine bacteria different sistematichnostiyu to the claimed phages are the stages of receipt, consisting in the allocation and accumulation of phage particles, verify the specificity of the properties and use of laboratory diagnostics. However, the known bacteriophages may not be used to identify and faguibine Legionella due to the high specificity of the mechanisms of interaction between bacteria and parasitic virus. For this reason they are not able to adsorb on the surface of cells Legionella, reproduce them and cause their lysis.

The specific strains legionellas of bacteriophages in the scientific and patent literature are not described.

The task of the invention to provide a specific strain regionalnego bacteriophage that is used to identify the main types of pathogens of Legionnaire's disease and laboratory diagnosis of legionellosis.

The solution is achieved by selection of the bacteriophage of the organs of laboratory animals infected with typical strain Philadelphia-1 Legionella pneumophila.

To isolate a homogeneous population of phage were negative selection of colonies. Pure line phage scientifically was obtained by five-time passage of separate negative colonies on the strain Philadelphia-1 L. pneumophila.

The strain of bacteriophage scientifically is stored in to the mi morphological and physiological properties.

Phage scientifically refers to the third morphological group of viruses, bacteria A. C. Tikhonenko (1968). The phage particles consist of multi-faceted elongated head, in the projection of the elongated hexagonal shape, size 600300and short ridge length 150±50(Fig.1-3). The type of nucleic acid - DNA molecular weight - 13106Yes.

Negative colonies formed layer BCYEa-agar, prepared according to the method of Grazia, with a typical indicator culture of L. pneumophila strain Philadelphia-1. After 72-96 hours of incubation at 35 ° -37Formed transparent colonies rounded shape with a relatively smooth edge, 1.5-2.5 mm in diameter. Application ragovoy suspension on the lawn of the indicator culture according to the method of Otto calls within 48-72 hours of incubation at 35 ° -37With the formation of a clear zone of specific lysis.

Adsorption of phage indicator bacteria in liquid medium containing 15 g/l of proteinopathy No. 3, 10 g/l yeast extract, 4 g/l To2NRA41 g/l KN2RHO4that 0,042 g/l Panso3, 0.25 g/l of pyrophosphate iron, 0.4 g/l of L-cysteine, the rest is di S. 208), is 90%, the latent period is 2 hours, the yield is 175 particles on infected cell.

When sowing dose n106microbial cells of 3-day broth cultures in 1 cm3the specified environment and multiplicity of infection of 0.1 for 24 hours incubation at 35 ° -37To accumulate up to n108FIGHTcm-3.

Phage thermolabile. Inactivation begins at a temperature of 50With, and complete destruction occurs after incubation at a temperature of 55C for 30 minutes.

Antigenic properties

After immunization of rabbits with the suspension of phage with Freud's adjuvant generated specific antibodies. The resulting anticavity neutralizes the phage with constant neutralization 43.

Specificity

The bacteriophage of vsokospecificno and analyzes the main types of bacteria pathogens legionellosis: L. pneumophila (9 strains), L. micdadei, L. dumoffii, and L. bozemanii. Phage scientifically has no political action on Yersinia pestis (5 strains), Vibrio cholerae, Francisella tularensis, Bacillus anthracis, Brucella abortus, Y. pseudotuberculosis, Escherichia coli (4 strains), Salmonella choleraesuis. Streptococcus faecium, equi, pneumoniae, Staphylococcus saprophyticus and aureus, Micrococcus luteus, Bacillus subtilis, Proteus vulgaris and mirabilis,sterowanie uranylacetate X 120000.

In Fig.2 shows the adsorption of bacteriophage particles scientifically on the surface of L. pneumophila. The contrast uranylacetate X 30000.

In Fig.3 shows the phage scientifically within the cells of L. pneumophila. The contrast uranylacetate X 50000.

Example. Phage scientifically reproducerea on cells of strain Philadelphia-1 L. pneumophila in a medium containing 15 g/l of proteinopathy No. 3, 10 g/l yeast extract, 4 g/l To2NRA41 g/l KN2RHO4that 0,042 g/l Panso3, 0.25 g/l of pyrophosphate iron, 0.4 g/l of L-cysteine, and the rest distilled water.

When planting a dose of 2106cells of the causative agent of legionellosis on 1 cm3environment and multiplicity of infection of 0.1 by incubation for 24 hours at a temperature of 35-37With up to n108phage particles on 1 cm3.

To obtain a preparation of phage scientifically 48-hour culture of strain Philadelphia-1 grown on medium BCYEa-agar, bring in a liquid nutrient medium from the calculation of the final concentration 5106cells on 1 cm3; after 48-72 hours rearing at a temperature of 35-37With and achieve a culture of concentration 2108cells on 1 cm3add FA the art of chloroform by volume). PageList added chloroform withstand 1.5-2 hours at room temperature (18-22(C) and 12 hours in the refrigerator (4-6C). Then PageList filtered through sterile filters with a pore size of 0.2 μm. The obtained filtrate is poured into vials and control specific, General sterility and activity.

Phage scientifically, like the typical phages other species used for faguibine main types of causative agents of Legionnaires in differentiating the working title (CES).

CES phage is determined as follows: the 48-hour culture of the causative agent of legionellosis strain Philadelphia-1 grown on medium BCYEa-agar, in the amount of 0.1 cm3with a concentration of 1.0 billion bacterial cells on industry standard turbidity CT, contribute in tubes with 4.5 cm3melted and cooled to 47From 0.7% BCYEa-agar (pH of 6.95), add in the amount of 0.1 cm3a tenfold dilution of phage scientifically. The contents of the tube mixed and poured onto the surface of agar plates (1.5% of BCYEa-agar, pH to 6.95). After solidification of the second layer crops incubated at 35-37C for 3-6 days. For CES accept that breeding, in which the phage scientifically forms of NCAC and definition differentiating titer.

Claims

Strain regionalnego bacteriophage scientifically possessing activity against Legionella pneumophila, L. micdadei, L. dumoffii, and L. bozemanii.

 

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9 cl, 5 tbl, 7 ex

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