RU2352633C1 - Strain of tularemia bacteriophage "+lt" - Google Patents

Abstract

FIELD: microbiology, medicine.

SUBSTANCE: strain of tularemia bacteriophage "ГАЛ" is intended for identification of main subspecies of Francisella tularensis and lyses tularemia causative agents of three subspecies (Middle Asian, Non-Arctic and Holarctic) and of main types of Legionnaires' disease, as well as for laboratory diagnistics of tularemia. Strain is isolated from organs of laboratory animals, infected with live culture of tularemia vaccine strain No 15 of Institute for Scientific Research of Epidemiology and Hygiene line. Bacteriophage strain is deposited in phage collection of Federal State Institution of "48 Central Institute for Scientific Research Departament of defence of Russia" under designation "ГАЛ".

EFFECT: increasing specificity of identification of tularemia causative agent from other causative agents.

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Description

The invention relates to medical microbiology, and in particular to means of laboratory diagnosis of tularemia, and can be used in scientific and practical laboratories involved in the indication and identification of tularemia bacteria secreted from the patient’s body and infected environmental objects.

Many bacteriophages are known that are used for identification and phagotyping of bacteria of various systematic positions (Medical Microbiology. / Ch. Ed. V.I. Pokrovsky, O.K. Pozdeev. - M .: GEOTAR, Medicine, 1999). Common to the claimed phages are the stages of their preparation, consisting in the isolation and accumulation of phage particles, verification of the specificity of properties and use for laboratory diagnosis. However, well-known bacteriophages cannot be used for identification and phagotyping of Francisella tularensis due to the high specificity of the mechanisms of interaction between bacteria and the parasitic viruses in them. For this reason, they are not able to adsorb on the surface of Francisella tularensis cells, reproduce in them and cause their lysis.

The existence of the phenomenon of bacterophagy in a tularemia microbe is known from the data cited in the literature (N.G. Olsufiev Taxonomy, Microbiology and Laboratory Diagnosis of the Tularemia Pathogen. - M., Medicine, 1975. - P.115-116). It was also noted there that the detected lytic agent belongs to moderate phages, however, it was not possible to isolate the bacteriophage.

Strains of specific tularemia bacteriophages are not described in the patent literature.

The objective of the invention is to obtain a specific strain of tularemia bacteriophage, designed to identify the main subspecies of Francisella tularensis and laboratory diagnosis of tularemia.

The solution to the problem is achieved by isolating the bacteriophage from the organs of laboratory animals infected with a live culture of tularemia vaccine strain No. 15 of the NIIEG line.

In order to isolate a homogeneous phage population, selection of negative colonies was carried out. A clean GAL phage line was obtained by ten-fold passaging of individual negative colonies on strain No. 15 of the NIIEG line of Francisella tularensis.

The strain of the bacteriophage GAL is stored in the phage collection of the Federal State Institution “48 Central Research Institute of the Ministry of Defense of Russia” (Kirov) and is characterized by the following morphological and physiological properties.

The phage GAL belongs to the first morphological group of bacterial viruses according to A.S. Tikhonenko (1968). The bacteriophage is a filamentous corpuscle with a thickness of 20 ± 4 nm and a length of up to 400 nm.

Negative colonies are formed on a two-layer FT-agar prepared according to the method of Grazia, with a typical indicator culture Francisella tularensis strain No. 501. After 36-72 hours of incubation at a temperature of 36-38 ° C, cloudy colonies form, as well as with a transparent center and an incomplete lysis zone at the periphery, the diameter of which does not exceed 1-2 mm. Turbid negative colonies have traces of the growth of indicator culture. Application of the phage suspension to the indicator culture lawn by the Otto method results in temperature control after 36-72 hours at 36-38 ° С, the formation of a specific lysis zone in the form of a “sterile” spot or a clearly contoured cloudy spot, or a group of small negative spots.

Phage adsorption by indicator bacteria in a liquid nutrient medium based on an enzymatic meat hydrolyzate diluted with distilled water to an amine nitrogen content of 145-155 mg% · dm ^-3 and with the addition of NaCl - 0.5 g; Na ₂ HPO ₄ · 12H ₂ O - 1.0 g; KCl - 0.2 g; yeast extract - 0.25 g ;, L-cysteine - 0.4 g; the rest is distilled water, at least 90 percent, the latent period is 4 hours, and productivity is 10-12 corpuscles per infected cell.

When a seeding dose of n · 10 ⁶ microbial cells of a 1-day agar culture in 1 cm ^{3 of the} specified medium and the multiplicity of infection of 0.01-0.001 within 24-36 hours of incubation at a temperature of 36-38 ° C accumulate to n · 10 ⁴ PFU · cm ^-3 .

The phage is thermolabile. Inactivation begins at a temperature of 50 ° C, and complete destruction occurs after incubation at a temperature of 55 ° C for 30 minutes.

Antigenic properties

When rabbits are immunized with a suspension of phage with Freund's adjuvant, specific antibodies are formed. The resulting antiserum neutralizes the phage with a neutralization constant of 51. The bacteriophage tularemia GAL refers to DNA-containing phages.

Specificity

The bacteriophage lyses the bacteria of tularemia pathogens of three subspecies (Central Asian, non-Arctic and Holarctic) and the main types of Legionnaire’s disease (Legionella pneumophila, Legionella, micdadei, Legionella dumoffii and Legionella bozemanii). The GAL phage has no lytic effect on the closely related species of Francisella novicida, as well as on Pasteurella multocida, Yersinia pestis (5 strains), Vibrio cholerae, Bacillus anthracis, Brucella abortus, Yersinia pseudotuberculosis, Escherichia coli (4 strain, strain strain) equi, Streptococcus pneumoniae, Staphylococcus saprophyticus and Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Proteus vulgaris and Proteus mirabilis, Shigella son-nei and Shigella flexneri, Serratia marii lecherineria legerminola gioliceria legermini

Figure 1 shows particles of tularemia bacteriophage GAL. Contrasting with uranyl acetate X 30000.

Figure 2 shows the adsorption of particles of the bacteriophage GAL on the surface of Francisella tularensis. Contrasting with uranyl acetate X 30000.

Example. GAL phage is reproduced on cells of Francisella tularensis strain No. 501 in a medium based on an enzymatic meat hydrolyzate with an amine nitrogen content of 145-155 mg% · dm ^-3 and 0.5 g of NaCl added; Na ₂ HPO ₄ · 12H ₂ O - 1.0 g; KCl - 0.2 g; yeast extract - 0.25 g; L-cysteine - 0.4 g; the rest is distilled water.

When a seed dose of 2 · 10 ⁶ cells of the causative agent of tularemia per 1 cm ³ medium and the multiplicity of infection of 0.01-0.001 when incubated for 24-36 hours at a temperature of 36-38 ° C accumulates up to n · 10 ⁴ phage particles per 1 cm ³ .

To obtain the preparation of GAL phage, a 24-hour culture of tularemia microbe strain No. 501 grown on FT-agar is introduced into a liquid nutrient medium to a final concentration of 5 · 10 ⁶ cells per 1 cm ³ , GAL phage is added (infection multiplicity 0.01-0.001 ) After 24 hours of incubation at a temperature of 36-38 ° C, chloroform by volume (1/35 part of chloroform by volume) is added to the phagolysate, incubated for 1.5-2 hours at room temperature (18-22 ° C) and 12 hours in a refrigerator ( 2-8 ° C). Then the phagolysate is filtered through sterile filters with a pore size of 0.2 μm. The resulting filtrate is poured into ampoules and monitored for specific, general sterility and activity.

GAL phage, like typical phages of other species, is used for phagotyping the main subspecies of the tularemia pathogen in a differentiating working titer (DRT).

The phage DDT is determined as follows: a 48-hour tularemia microbe of strain No. 501 grown on FT-agar in a volume of 0.1 cm ³ with a concentration of 1.0 billion bacterial cells according to the industry standard turbidity GKI turbulence is added to 10 tubes with 4.5 cm ³ molten and cooled to 47 ° C. 0.7% FT-agar (pH 6.95). Then, ten-fold dilutions of the phage of GAL are successively added to the tubes in a volume of 0.1 cm ³ . The contents of the tubes are mixed and poured onto the surface of agar plates (1.5% FT-agar, pH 6.95). After the second layer has hardened, the crops are incubated at 36-38 ° C for 2-4 days. A dilution in which the GAL phage forms at least 10 negative colonies is taken as a DRT.

The sensitivity of the studied cultures to the GAL phage is studied in the same way as the determination of the differentiating working titer.

Claims (1)

The strain of tularemia bacteriophage GAL-KSM No. 17-4 (Collection of standardized microorganisms to test the effectiveness of intelligence, prevention and protection against infectious pathogens FSI "48 Central Research Institute of the Russian Ministry of Defense"), which has specific lytic activity against cultures of strains of Francisella tularensis and pathogens of the main species Legionnaire's disease.

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