RU2425877C1 - BACTERIOPHAGE Escherichia coli V32 STRAIN FOR IDENTIFICATION OF Escherichia coli BACTERIA SEROGROUP O157 - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: bacteriophage Escherichia coli V32 strain is deposited in the Collections of Museum of microorganisms of Federal State Intuition of Science State Science Centre of Applied Microbiology and Biotechnology, No. Ph25 is specific and virulent for bacteriophage, and used as an option for identification of Escherichia coli bacteria serogroup O157 at the stage of bacterial colony analysis. ^ EFFECT: simple and available product not requiring heavy time and material consumption. ^ 1 dwg, 4 ex

Description

The invention relates to medical microbiology and can be used for epidemic institutions as an additional tool for the identification of bacteria Escherichia coli serogroup O157.

In recent years, there have been a large number of reports of outbreaks in humans of hemorrhagic gastroenteritis caused by bacteria Escherichia coli O157. The solution to the problem largely depends on the timely diagnosis of the pathogen. In Russia, there are no commercial preparations for the detection of these pathogens.

Known nutrient medium (1), which is semi-selective for the isolation of sorbitol-negative colonies of Escherichia coli from clinical material and food products. To identify among them the bacteria Escherichia coli serogroup O157, additional studies are required.

A kit for the identification of enterohemorrhagic Escherichia (2), consisting of an immunomagnetic separation test system and a test system for setting up a multiplex polymerase chain reaction (PCR), is also proposed. The set has high specificity and sensitivity. However, its use requires special equipment and trained personnel. In addition, in some cases, ambiguous results are obtained related to the wide distribution in nature of genes used for testing.

Abroad, three methods are usually used to detect E. coli O157: microbiological, PCR, and immunoassays. In addition, a labeled recombinant bacteriophage PPO1 was proposed, which allows the detection of E. coli O157 bacteria within 10 minutes in the presence of a fluorescence microscope (3).

The technical result of the invention is the use of highly specific virulent bacteriophage Escherichia coli V32 for identification of E. coli O157 bacteria.

The strain of the bacteriophage Escherichia coli V32 was isolated from the effluent of a barn located in the Moscow region and was deposited in the collection of the Museum of Microorganisms of the Federal State Institution of Science and Science of the Center for Applied Microbiology under the number Ph25.

The Escherichia coli V32 bacteriophage strain is recommended to be used at the stage of analysis of suspicious sorbitol-negative colonies of microorganisms grown on MacConkey medium with sorbitol or on medium (1).

The use of such a bacteriophage will make it easy to identify Escherichia coli serogroup O157 bacteria without the involvement of special equipment and personnel training.

The bacteriophage strain Escherichia coli V32 is characterized by the following properties:

- morphology of negative colonies: on a sensitive bacterial strain forms transparent negative colonies with a diameter of about 2-3 mm, with a clear edge. Secondary growth is absent;

- refers to the lambdoid group of phages;

- virulent for 100% E. coli O157 bacteria used for testing;

- specific for bacteria E. coli O157. Not active for other serotypes of Escherichia, Shigella and Yersinia species;

- does not contain pathogenicity factors of E. coli O157: H7 (rfb, stxl, stx2, еае and fliC);

- does not form stable lysogens;

- the genome has double-stranded DNA about 62 thousand base pairs (kb) in size. DNA is not hydrolyzed by SalG1, EcoRI, BamHI, PstI enzymes. HindIII endonuclease cleaves DNA into 28 fragments, EcoRV into 13.

- storage in the form of a phagolysate, or in a lyophilized state.

Example 1. Obtaining a phagolysate strain of the bacteriophage Escherichia coli V32.

To 1 ml of night broth culture of E. coli O157: H7 EDL933 add 4 ml of Luria broth and 1 μl of a suspension of the bacteriophage strain Escherichia coli V32 with a concentration of 5 × 10 ⁹ PFU / ml. The mixture is incubated on a rocking chair at 37 ° C until the onset of enlightenment of the bacterial culture. Add 0.5 ml of chloroform and mix vigorously in a mixer for 20 minutes. Low-speed centrifugation (10000g, 15 min) removes fragments of bacterial cells and titrates the phagolysate. The resulting phagolysate contains more than 10 ⁹ phage particles in 1 ml.

Example 2. Verification of the specificity of the bacteriophage strain Escherichia coli V32.

The specificity of the bacteriophage strain Escherichia coli V32 is checked by the method of spot tests. To do this, 0.5 ml of the nightly broth bacterial cultures indicated in Table 1 are mixed with 4 ml of semi-liquid agar (Luria broth with 0.7% agar) at a temperature of 47 ° C and distributed over the surface of a Petri dish containing a rich nutrient medium (e.g. Nutrient Agar, HIMEDIA). After 20 minutes, a drop of the bacteriophage V32 strain (about 10 ⁷ PFU / ml) was applied to the surface and incubated at 37 ° C overnight. On visual inspection, only on plates with cultures of E. coli O157: H7 and E. coli O157, bacterial lawn growth is absent at the sites of the introduction of the bacteriophage strain Escherichia coli V32 (transparent lysis spot). In all other cases, such zones are not detected.

Table 1 The specificity of the bacteriophage strain Escherichia coli V32 (spot tests). Bacterial strains Lysis spot Escherichia coli O157: H7 (60 *) + O157: H ^- (8 *) + Other serotypes of E. Coli (26 **) - Salmonella cholerae suis (15 *) - gallinarum pullorum (9 *) - typhimurium (2 *) - dublin (1 *) - enteritidis (3 *) - Shigella dysenteriae (1 *) - Shigella sonnei (1 *) - Yersinia enterocolitica (3 *) - pseudotuberculosis (4 *) - In parentheses: *, the number of strains of this species used in testing;
**, E. coli strains of serotypes O1, O2, O4, O6, O8, O15, O18, O19, O20, O23, O26, O28, O29, O33, O55, O78, O79, O110, O111, O113, O117, O119, O127, O139, O140 and O142;
"+", Lysis; “-”, no lysis.

Example 3. Enzymatic hydrolysis of DNA of the bacteriophage strain Escherichia coli V32.

A restriction mixture is prepared containing 0.1 μg / ml DNA of the bacteriophage strain Escherichia coli V32, 1 μl of 10-fold restriction buffer, 1 unit of the corresponding enzyme, deionized water to a total volume of 10 μl. The mixture is incubated at a temperature of 37 ° C for 1 hour. Electrophoresis is carried out in a horizontal electrophoresis apparatus at a constant current of 150 mA for 1 hour. After 15 min of staining in a solution of 0.4 μg of ethidium bromide, the gel is visualized under a UV lamp (see drawing).

Example 4. The use of a strain of the bacteriophage Escherichia coli V32 for the analysis of bacterial colonies using the spot test.

Bacterial colonies of the analyzed bacteria are individually suspended in 0.1 ml of sterile saline and transferred to plates with Nutrient Agar medium. Suspension to dryness is rubbed with a sterile spatula into the surface of the agar and, after dividing the cup into 2 sectors, a drop of the bacteriophage strain Escherichia coli V32 is pipetted into one of them and a drop of physiological saline into the other. The plates are incubated overnight at 37 ° C. On visual inspection, only on plates with Escherichia coli V32 phage sensitive bacterial cultures (E. coli O157: H7 or E. coli O157: H ^- ) there is no bacterial lawn growth at the site of bacteriophage insertion. In all other cases, there are no such zones.

Example 5. The use of a strain of the bacteriophage Escherichia coli V32 for the analysis of bacterial colonies by the phage "track" method.

A drop of the bacteriophage strain Escherichia coli V32 is applied to the surface of Nutrient Agar and, by tilting the Petri dish, distribute it in diameter. The phage "track" is dried. To do this, the cup, having opened the lid, is placed for 15 min in a thermostat at 37 ° C. After this, the suspension of colonies of the analyzed bacteria is transferred by a bacteriological loop to the surface of the nutrient agar so that they cross the phage "path" perpendicularly. Cups are incubated overnight at a temperature of 37 ° C. By visual inspection only culture of E. coli O157: H7 and E. coli O157: H ^- growing discontinuous strip due to the lack of growth in the area of phage "track". The rest of the cultures grow in a continuous continuous strip.

Thus, the bacteriophage strain Escherichia coli V32 is specific and virulent for bacteria Escherichia coli serogroup O157. The use of the proposed bacteriophage strain for identification of bacteria of Escherichia coli serogroup O157 is a simple and generally available method that does not require large expenditures of time and materials.

Information sources

1. Patent RU No. 2139343, C12Q 1/20. Bull. No. 28, 1999

2. Patent RU No. 2270253, C12Q 1/68. Bull. No. 5, 2006

3. Oda M., Morita M., Unno N., Tanji Y. Applied Environmental Microbiol. 2004; 70: 1: 527-534.

Claims (1)

The strain of the bacteriophage Escherichia coli V32 for the identification of bacteria Escherichia coli serogroup O157, deposited in the collection of the Museum of microorganisms FSHI SSC PMB under the number Ph25.

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