RU1825375C - "yersinia pestis" pestilential microbe strain used as test object for determining constitution and function of new additional pestilential pathogen plasmide - Google Patents

Abstract

Usage: biotechnology, medical microbiologists, genetics of microorganisms. The essence of the invention: obtaining a new strain of plague microbe Yerslnla pestls having an additional plasmid mol. m. 20 mDa. The strain was isolated from fleas of a large gerbil, caught 120 km northeast of the village of Bakanas. It has cultural-morphological and other properties typical of the plague pathogen and can be used as a genetic probe in the identification and detection of strains of the plague pathogen isolated from various natural foci.

Description

The invention relates to biotechnology and can be used in medical and research microbiology.

The strain of the plague microbe Yerslnla pestls A-1567 was isolated from fleas of a large gerbil caught 120 km northeast of the village of Bakanas. The strain is obtained by direct plating of biological material on agar plates of Hottinger medium at 28 ° C. On the second day of incubation, a typical growth of the plague pathogen is noted.

Strain A-1567 is characterized by the following features.

Cultural and morphological characteristics: strain A-1567 has cultural morphological and other properties typical of the plague pathogen. It grows in the R-form on ordinary nutrient media (Hottinger broth and agar, Marten medium, ordinary nutrient agar, Endo medium, yeast medium, casein and gelatin hydrolysates).

The diameter of the colonies with isolated growth on Hottinger medium for 2–3 days is 2–3 mm, and for the fifth, 2–8 mm (at 28 ° C cultivation).

The optimum growth temperature is 27-28 ° С, minimum (barely noticeable growth on the 3rd day) - + 2 ° С, maximum - + 40 ° С. On the Jackson-Burroughs medium, it grows in the form of populations consisting of pigmented (pgm +) colonies. On liquid culture media, the growth pattern is typical of a plague microbe. Gram-negative bipolar bacilli appear in smears.

The nutritional needs of the strain. The strain grows at 28 ° C on a medium of the following composition, g / l; glucose 3;, disubstituted potassium phosphate 7; monosubstituted potassium phosphate 2; sodium citrate 0.5; magnesia sulfate 0.1; sodium sulfite 0.5; cysteine 0.05; phenylalanine 0.05; methionine 0.05; threonine 0.05 (amino acids in L- and DL-form), distilled water 1

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l (in the case of solid culture media, 30 g of agar-agar is added).

Physiological and biochemical characteristics. Fermented with the formation of acid without gas, glucose, maltose, mannitol, arabinose, mannose, xylose, glycerin. It does not ferment lactose, sucrose, rhamnose. dulcite, erythritis, melibiosa cellobinoza. It gives negative reactions of denitrification and nitrification. Has / -galactosidase. The Voges-Proskauer reaction is positive. It does not have the enzyme urease, oxidase, reductase tetrathionate, amylase, decarboxylase lysine and arginine dehydrolase. Gelatin does not dilute or break down sodium malonate and sodium tartrate. Calcium Dependent. In Higuchi-Smith medium, it mutates into calcium-independent cells fCa4} with a low frequency (2-8 x 10 4-7-8 x).

Relation to homo- and heterologous strains. Highly sensitive to diagnostic plague and pseudotuberculosis phages and phage L-413 C.

The ratio of strain to species diagnostic sera. Agglutinated with anti-plague serum at a titer of 1: 1200.

Antigenic properties of the strain. It has fra, ina, and Tox antigens.

Sensitivity to antibiotics. It is highly sensitive to streptomycin, rifampicin, gentamicin, penicillin, monomycin, tetracycline, chloramfericol, chlortetracycline.

Genetic features of the strain. Strain auxotrofen. It has the plasmids pPst, pCad (pVIr), pFra Tox and the additional plasmid mole, which are common for the plague pathogen m. 20 mDa.

Virulent properties. Strain A-1567 is highly virulent for laboratory animals — guinea pigs die from 10 and white mice from 102 microbial cells by subcutaneous infection.

Storage conditions. The strain is stored in sealed tubes on the braids of the Hottinger medium in a refrigerator (3-10 ° C).

Conditions for identifying strain products of the strain. Hottinger medium (incubation temperature of 37 ° C) can be used to detect toxin and antigenic fraction 1. Jackson-Burrows medium is used in the usual recipe to detect pigment absorption.

Example. The culture of the strain and Escherichia coli was grown during the day on

Hottinger agar (pH 7.2), then cells were removed from an area of 1 cm2 and suspended in 200 ml of buffer containing 50 mM Tris, 2.5% sodium dodecyl sulfate and NaOH (to pH 12.5). Samples were placed in a water bath at 68 ° С for 30 min. An equal volume of phenol-chloroform (1: 1) was added and mixed thoroughly. Centrifuged for 5 min at 15 thousand, rpm The aqueous phase is mixed with 1/10 of the volume of the buffer and applied to the wells of a 0.6% agarose gel. Electrophoresis was carried out in horizontal gel plates in TBE buffer: 69 mM Tris, 89 mM NhBOz, 2.5 mM Na2 - EDTA. DC voltage 100 V, duration

5 electrophoresis of 3.5 hours. As markers, mol. m. E. coli plasmids are used: pCRI (7.5 MDa), pRK290 (13 mDa), pSa (23 mDa), R6K (26 mDa), RP4 (37 mDa). As a result of the experiment, the presence of an additional plasmid in strain A-1567 was revealed. Like m of the plasmid is 20 mDa.

Thus, a new strain of the plague pathogen containing an additional plasmid mol. m. 20 mDa, can be used as a genetic probe in the identification and detection of strains of the plague pathogen isolated from various natural foci.

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Claims (1)

The claims The plague microbe strain Yerslnla Pestls KM-939, used as a test object to determine the structure and function of a new additional plasmid pathogen plasmid.