RU2083663C1 - STRAIN OF BACTERIUM KLEBSIELLA PNEUMONIAE-232 - A PRODUCER OF β --HEMOLYSIN - Google Patents

Abstract

FIELD: microbiology, biotechnology. SUBSTANCE: invention relates to new strain of bacterium Klebsiella pneumoniae showing stable property to produce β --hemolysin. Strain is isolated from patient with diarrhea and not typified. Strain is deposited at N 232 in GISK Collection and can be used for preparing diagnostic preparations and as reference-strain for study of β --hemolytic activity of strains. EFFECT: new strain indicated above. 1 tbl

Description

 The invention relates to the field of medical microbiology, namely to biotechnology, and can be used in the technological process for the production of β-hemolysin to obtain diagnostic products, as well as a reference strain in the study of b-hemolytic activity of strains.

 The purpose of the invention is the creation of a strain stably producing b-hemolysin, the use of which simplifies the method of producing b-hemolysin as an antigenic drug.

 The strain Klebsiella pneumoniae was isolated from clinical material with a patient with diarrhea, and was deposited in the collection of the State Research Institute for Standardization and Control of Medical and Biological Preparations named after L.A. Tarasevich under N 232.

 Characterization of the strain. Cultural and morphological signs: Gram stain: gram-negative rods with rounded ends. They do not form a dispute.

 Coloring in a carcass preparation according to Burri: gram-negative small sticks with a distinct capsule, the size of which reaches 1/2 the diameter of the body of the bacterial cell.

 Growth on Endo medium: bright pink colonies with a darker color in the center, convex, oily, the diameter of the colonies is 1.5-2.0 mm. There are no signs of dissociation.

Growth on K ₂ medium: light yellow colony, oily, colony diameter 2-3 mm, high with smooth edges. There are no signs of dissociation.

 Growth on meat-peptone agar: colonies are light, translucent, oily with smooth edges, diameter 1.0-1.5 mm. There are no signs of dissociation.

 Growth on meat-peptone broth: cause a uniform turbidity of the medium, with a slight swaying of the crusher of the appearance of moire waves.

Physiological and biochemical characteristics: optimum growth at a temperature of (37 ± 1) ^o C, optimum pH of the medium 7.0-7.2, optimal aerobic conditions. Actively breaks down glucose and glycerin with the formation of acid and gas, and also breaks down lactose, beckons, sucrose, maltose, arabinose, adonite, inositol, dulcite, xylose, sorbitol, rhamnose. It has urease activity. It gives a positive reaction to indole and Voges-Proskauer. It utilizes Simons citrate, Coser citrate and sodium malonate. Gives a negative reaction to hydrogen sulfide and methyl-mouth. Lysine decarboxylase is present. Ornithine decarboxylase and arginine dehydrolase are absent.

Strain K. pneumoniae 232 non-typable. Serotyping is carried out with a set of K ^- serum UNIViS or Mechnikov.

 Sensitivity to antibacterial drugs.

 Antibiotic resistance is determined by serial dilution.

 It was found that the strain is resistant to benzylpenicillin (10 μg / ml) ampicillin (1.5 μg / ml), ampiox (1.5 μg / ml), tetracycline (1.5 μg / ml), doxycycline (30 μg / ml) .

 The strain is sensitive to neomycin (1.0 μg / ml), kanamycin (5.0 μg / ml), gentamicin (2.5 μg / ml), sisomycin (1.0 μg / ml), kefzola (1.0 μg / ml), claforan (1.0 μg / ml), cephalotin (1.5 μg / ml), as well as levomycetin (5 μg / ml), polymyxin (2.5 μg / ml), nalidixic acid (25 μg / ml) and trimethoprim (25 μg / ml).

Strain activity
The intensity of production of b-hemolysin is determined by the zone of clarification of the nutrient medium containing red blood cells after 18-24 hours of cultivation at a temperature of (37 ± 1) ^o C. The lysis zone is 4-5 mm. The strain lyses sheep red blood cells.

Virulence in the model of a white mouse: LD ₅₀ 2 • 10 ⁹ mt

 The strain is used as follows.

Example 1. The strain culture is seeded by depletion of the loop in Petri dishes with meat-peptone agar containing 5 suspensions of sheep erythrocytes. The Petri dish is maintained at a temperature of (37 ± 1) ^o C for 18-24 hours. At the end of the specified period, an inventory is made. The enlightenment zone is 3-4 mm. Then the Petri dish with crops is kept at a temperature of (6 ± 4) ^o C for 2-3 hours and taken into account again, the enlightenment zone is 5-6 mm around each colony.

Example 2. The strain culture is seeded on beveled meat-peptone agar and grown at a temperature of (37 ± 1) ^o C for 18-24 hours and rinse with 5 ml of sterile saline. A suspension of 10 ⁹ mt / ml, 0.5 • 10 ⁹ mt / ml, 1 • 10 ⁸ mt / ml; 0.5 • 10 ⁸ mt / ml; 1 • 10 ⁷ mt / ml; 0.5 • 10 ⁷ mt / ml.

From a sheep’s saline erythrocyte washed with physiological solution, 3 suspensions are prepared and 1 ml of a number of wells of titration plates are added. Then, 1 ml of the culture cultivated by two-step dilution is successively added. The tablet is closed with glass and kept at a temperature of (37 ± 1) ^o C for 1 hour. After the specified period, take into account. Complete hemolysis of sheep erythrocytes is observed at a culture concentration of 1 • 10 ⁷ bw / ml.

Example 3. Wash off the daily agar culture obtained as described in example 2, and subjected to ultrasonic disintegration at an amplitude of 15-16 kHz for 7-10 minutes at a temperature of minus 1-0 ^o C. Debris of bacterial cells precipitated by centrifugation at 8000 rpm for 30 min at a temperature of (6 ± 4) ^o C. The supernatant is subjected to research, pre-triturated in physiological solution with two-step dilution 1: 2, 1: 4, etc. up to 1: 128.

 In an agar containing 5% suspension of sheep erythrocytes, 4 mm diameter wells are punched and 25-30 μl of the grown supernatant are added and kept at room temperature for 4-6 hours. The lysis zone at a 1:64 dilution of 5-6 mm.

Example 4. The study of the ability of strain K. pneumoniae 232 to stably produce b-hemolysins was carried out as follows: the strain was stored on semi-liquid hungry agar under a layer of liquid paraffin at a temperature of (6 ± 4) ^o C and freeze-dried every 6 months. subjected to research, as described in example 1.

 As can be seen from the table, the strain K. pneumoniae N 232 has the ability to produce b-hemolysin.

 Thus, the strain K. pneumoniae N 232 is a stable producer of b-hemolysin, which can be used in the production of hemolysin as an antigen necessary for the production of diagnostic drugs, as well as a reference strain for studying the hemolytic properties of Klebsiella and other enterobacteria cultures.

Claims (1)

 The bacterial strain Klebsiella pneumoniae GISK N 232 producer β - hemolysin.

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