RU2496874C1 - STRAIN OF BACTERIOPHAGE Escherichia coli ECD4, HAVING LYTIC ACTIVITY IN RESPECT TO BACTERIA Escherichia coli OF SEROTYPE O104:H4 - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention relates to a strain of the bacteriophage Escherichia coli ECD4. The strain of the bacteriophage Escherichia coli ECD4 is isolated from faeces of broiler chickens on the culture of the bacteria of the strain Escherichia coli O104.H4 RK1No.112027 and is deposited in the State Collection of Pathogenic Microorganisms and Cell Cultures "GKPM-Obolensk" under the number Ph63. The proposed strain of the bacteriophage has lytic activity in respect to the bacteria of the strain Escherichia coli O104:H4 RKINo.112027, lyses Escherichia of the serotype 0157:H7, does not suppress growth of cells Escherichia coli M-17, has lytic activity in respect to several other clinically significant serotypes of Escherichia, and also to several types of Shigella. The strain of the bacteriophage Escherichia coli ECD4 propagates on the laboratory non-pathogenic strain Escherichia coli K-12 C600F.

EFFECT: invention may be used in creation of new preparations for treatment of a disease caused by bacteria Escherichia coli and for elimination of this pathogen from food products.

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Description

The invention relates to medical microbiology and relates to a strain of the bacteriophage Escherichia coli ECD4, which can be used to create new drugs for the treatment of diseases caused by Escherichia coli bacteria of serotype O104.H4, as well as to eliminate this pathogen from food.

Some strains of Escherichia coli bacteria producing Shiga-like toxins cause outbreaks of hemorrhagic colitis (PS), accompanied by severe complications in the form of hemolytic uremic syndrome (HUS). HUS is characterized by acute renal failure, hemolytic anemia, thrombocytopenia. Mortality during the development of HUS is high and ranges from 3 to 30% [Hemolytic uremic syndrome and death in persons with Escherichia coli O157: H7 infection, foodborne diseases active surveillance network sites, 2000-2006 / L.H. Gould, L. Demma, T.F. Jones et al. - Clinical Infectious Diseases. - 2009 .-- Volume 49 (10). - P. 1480].

Most often, enterohemorrhagic Escherichia coli (EOL) of serotype O157. H7 are distinguished from patients with HUS, which are considered as the clinically most significant, however, other serogroups of Escherichia [Epidemiology of Escherichia coli O157: H7 outbreaks, are increasingly becoming causative agents United States, 1982-2002 / JMRangel, PHSparling, C. Crowe et al. - Emerging Infectious Diseases. - 2005. - Volume 11 (4). - P. 134-151]. In particular, in the spring and summer of 2011, an outbreak of hemorrhagic colitis with HUS was caused in Germany and other countries of the European Union, caused by bacteria of the “new” highly pathogenic strain Escherichia coli serotype O104: H4 producing a Shiga-like toxin type 2. This strain differs from the “classical” EHEC in the mechanism of adhesion and penetration of its toxin through intestinal epithelial cells. The treatment of hemorrhagic colitis caused by Escherichia serotype O104: H4 is seriously complicated by the fact that the cells of this strain have multiple antibiotic resistance [Characteristics of the enteroaggregative Shiga toxin / verotoxin-producing Escherichia coli O104: H4 strain causing the outbreak of haemolytic uraemic syndrome Germany, May to June 2011 / F.Scheutz, E. Nielsen, J. Freemodt, N. Boisen et al. - Eurosurveillance. - 2011. - Volume 16. - P. 134-145]. In this regard, there is a need to develop new drugs for the treatment of HA caused by Escherichia coli serotype O104: H4, and the elimination of these bacteria from food products. In this regard, bacteriophages may be useful.

A known bacteriophage strain Escherichia coli V32, capable of lysing enterohemorrhagic Escherichia serogroup O157, proposed for identification of bacteria of this serogroup [Patent RU 2425877, application 2010106806/10, 02.26.2010]. However, this phage is not able to lyse the bacteria Escherichia coli serotype O104: H4.

The technical result of the invention is to obtain a bacteriophage Escherichia coli ECD4, having lytic activity against Escherichia coli bacteria of serotype O104: H4 and suitable for creating a drug for the treatment of diseases caused by bacteria Escherichia coli O104: H4, as well as to eliminate this pathogen from food products .

The strain of the bacteriophage Escherichia coli ECD4 was isolated from broiler chicken feces on a bacterial culture of the strain Escherichia coli O104: H4 RKI No. 112027 and deposited in the State collection of pathogenic microorganisms and cell cultures GKPM-Obolensk under the number Ph 63.

The strain of the bacteriophage Escherichia coli ECD4 is characterized by the following properties:

- morphology of negative colonies: on a sensitive bacterial strain forms turbid negative colonies with a diameter of about 1 mm; secondary growth is absent;

- inhibits the growth of bacteria of the strain Escherichia coli O104: H4 RKI№112027 to the eighth ten-fold dilution when titrated according to Appelman;

- possesses lytic activity against Shiga-toxin-producing strains of Escherichia coli serotypes O104: H4 and O157: H7, as well as clinically significant Escherichia coli of other serogroups; in addition, the bacteriophage strain Escherichia coli ECD4 lyses bacteria of species such as Shigella sonnei and Shigella flexneri, the causative agents of dysentery in humans;

- does not inhibit the growth of bacteria non-pathogenic for humans strain Escherichia coli M-17, used for the preparation of the probiotic preparation "Kolibacterin"

- does not contain genes for the synthesis of Shiga-like toxins;

- no clones of the strain Escherichia coli O104: H4 RKI No. 112027 resistant to the bacteriophage Escherichia coli ECD4 were detected;

- the genome has double-stranded DNA about 60 thousand base pairs in size. DNA is not hydrolyzed by SalGI, PstI, EcoRI, BamHI, HindIII, ApaI, KpnI, Ecol47I, Eco52I, Ecol30I, MvaI enzymes. Endonuclease SmiI splits the DNA of the bacteriophage strain Escherichia coli ECD4 into three fragments, two of which are 700 and 3000 base pairs in size, BcuI splits DNA into six fragments, EcoRV - 15, XmiI - 18, VspI - 20 fragments.

- for propagation of the bacteriophage strain Escherichia coli ECD4, a non-Shiga toxin-containing laboratory non-pathogenic bacterial strain Escherichia coli K-12 C600F is used, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number B-7158.

The strain of the bacteriophage Escherichia coli ECD4 is stored as a phagolysate, or in a lyophilized state.

Example 1. Obtaining the phagolysate strain of the bacteriophage Escherichia coli ECD4.

To 1 ml of overnight broth culture of Escherichia coli K12 C600F add 4 ml of LB broth (per 1 liter of medium 10 g of Bacto tryptone, 5 g of Bacto yeast extract, 10 g of NaCl) and 10 μl of a suspension of the bacteriophage strain Escherichia coli ECD4 with a concentration of 10 ⁹ PFU / ml The mixture is incubated on a shaker at 180 rpm and a temperature of 37 ° C until the onset of bacterial culture enlightenment, add 0.5 ml of chloroform and mix vigorously for 20 minutes. Low-speed centrifugation (10,000 g, 15 min) removes the debris of bacterial cells and titrates the phagolysate using the double agar layer method. The concentration of the bacteriophage is expressed as the number of plaque forming units (PFU) in 1 ml of phagolysate. In the resulting phagolysate, it is 10 ⁹ PFU / ml.

Example 2. Verification of the lytic activity of the bacteriophage strain Escherichia coli ECD4 against bacteria of the strain Escherichia coli O104: H4 RKI No. 112027.

The lytic activity of the bacteriophage strain Escherichia coli ECD4 against bacteria of the strain Escherichia coli O104: H4 RKI No. 112027 is checked by the Appelman method. In tubes containing 4.5 ml of LB broth, ten-fold serial dilutions of Escherichia coli ECD4 bacteriophage strain are made and 5 μl of night broth culture of Escherichia coli strain O104: H4 RKI No. 112027 are added to each of them. In addition, two control tubes are prepared: in one control tube, only 5 μl of the overnight broth culture of the strain Escherichia coli O104: H4 RKI No. 112027 is added to 4.5 ml of LB broth in the second test tube, and only 0.5 ml of the bacteriophage strain Escherichia coli ECD4 is added to the second. The tubes are incubated in a thermostat at a temperature of 37 ° C for two days. The result of the study is evaluated visually. In all tubes up to the eighth ten-fold dilution of the bacteriophage, the medium remains transparent, as in a control tube containing only the bacteriophage strain Escherichia coli ECD4. In a test tube with a ninth ten-fold dilution, where the concentration of the bacteriophage is the lowest, and in a control tube containing only bacteria of the Escherichia coli strain O104: H4 RKI No. 112027, turbidity of the medium is observed.

Example 3. Enzymatic hydrolysis of DNA of a bacteriophage strain of Escherichia coli ECD4.

A restriction mixture is prepared containing 0.1 μg / ml DNA of the bacteriophage strain Escherichia coli ECD4, 1 unit of the required enzyme and 2 μl of 10-fold corresponding restriction buffer, deionized water to a total volume of 20 μl. The mixture is incubated at a temperature of 37 ° C (for Smil restrictase - 30 ° C) for 1 hour. Electrophoresis is carried out in a horizontal electrophoresis apparatus in Tris-borate buffer at an electric field strength of 8-10 volts / cm for 1 hour. After 15 min staining in an aqueous solution of ethidium bromide (0.4 μg / ml), the gel is visualized under a UV lamp. The result of enzymatic hydrolysis of DNA of the bacteriophage strain Escherichia coli ECD4 is illustrated in figure 1.

Figure 1 - DNA Hydrolysis of a strain of the bacteriophage Escherichia coli ECD4 restriction endonucleases BcuI (lane 1); SmilI (3); jointly SmiI and BcuI (4); 2 - native DNA of the bacteriophage strain Escherichia coli ECD4; M - lambda phage DNA cleaved with the HindIII endonuclease (molecular weight marker: from top to bottom - 23130, 9416, 6557, 4361, 2322, 2027, 564 base pairs).

Example 4. Evaluation of the spectrum of antibacterial action of a bacteriophage strain Escherichia coli ECD4.

The antibacterial spectrum of the bacteriophage strain Escherichia coli ECD4 is determined by the spot test method. For this, 0.5 ml of the nightly broth bacterial cultures indicated in Table 1 are mixed with 4 ml of semi-liquid agar (LB 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 strain Escherichia coli ECD4 (10 ⁷ PFU / ml) was applied to the surface and incubated in an incubator at a temperature of 37 ° C for 24 hours. The result of the study is evaluated visually by the presence or absence of lysis spots on the lawn of the culture at the place of application of a drop of a bacteriophage strain Escherichia coli ECD4.

Table 1 The lytic spectrum of the bacteriophage strain Escherichia coli ECD4 Bacterial strains Lytic 1 activity Escherichia coli O104: H4 RKI No. 112027 + Escherichia coli O157.H7 (8) * + (6) * - (2) Escherichia coli M-17 - Escherichia coli other serogroups: - O104: H12; + - O2; + - O1, O4, O79, O111, O115, O138, O139, O147; - - O6, O26, O78, O126; + - O25 (4); + (2) - (2) - O78 (6) + (2) - (4) Escherichia coli K-12 (5) + Shigella sonnei (2) + Shigella flexneri (17) + (11) - (6) Shigella dysenteriae -

* - in brackets - the number of strains of a given species, serogroup, serotype used during verification;

"+" - lysis; “-” - lack of lysis.

Thus, as can be seen from examples 2 and 4, the bacteriophage strain Escherichia coli ECD4 has lytic activity against bacteria of the epidemic strain Escherichia coli O104: H4 RKI # 112027, lyses Escherichia serotype O157: H7, does not inhibit cell growth of the non-pathogenic strain Escherichia coli M- 17 used for the preparation of the preparation "Kolibacterin" has lytic activity against other clinically significant Escherichia serogroups, as well as some Shigella species, presented in Table 1. The bacteriophage strain Escherichia coli ECD4 propagates in laboratory Amma Escherichia coli K-12 C600F, operating time so it is simple and does not require special conditions. The proposed strain of bacteriophage Escherichia coli ECD4 can be used to create drugs for the treatment of a disease caused by Escherichia coli bacteria of serotype O104: H4, as well as for their elimination from food.

Claims (1)

The strain of bacteriophage Escherichia coli ECD4, which has lytic activity against Escherichia coli bacteria of serotype O104: H4, was deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number Ph63.

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