RU2566555C1 - Burkholderia cepacia B-7518 STRAIN USED FOR OBTAINING OF ANTIGEN FOR IDENTIFICATION OF ANTIBODIES TO Burkholderia cepacia IN BIOLOGICAL ENVIRONMENTS - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: Burkholderia cepacia V-7518 strain with typical and stable cultural, tinctorial, biochemical properties is described.

EFFECT: strain can be used for obtaining of anti-gene for indentification of antibodies to Burkholderia cepacia in biological environments.

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Description

The invention relates to medical microbiology and biotechnology and relates to a strain of Burkholderia cepacia B-7518, which can be used to obtain antigen for the detection of antibodies to Burkholderia cepacia in biological media.

It is known that B. cepacia bacteria can cause pathological changes in humans. The most acute infection problem of B. cepacia is in patients with cystic fibrosis, as well as in connection with the high frequency of spread of acquired immunodeficiencies among the population. The problems of treating patients with B. cepacia infections are complicated by the high resistance of this microorganism to many antibiotics and the appearance of multiresistant clones. The search and development of environmentally friendly, harmless biological products for the treatment and prevention of this infection are relevant.

To obtain an antigen for the determination of antibodies to B. cepacia in biological media on an industrial scale, a strain of B. cepacia, which is standard in terms of growth, biochemical and antigenic characteristics, is required, which will ensure biomass growth while maintaining properties.

The aim of the invention is the selection of a strain of B. cepacia, standard in terms of growth, biochemical and antigenic characteristics, providing stably high growth when cultivated on solid and liquid nutrient media and preserving its properties.

The proposed strain of B. cepacia B-7518 has typical morphological, tinctorial, biochemical and cultural characteristics typical of B. cepacia. Cells are motile, gram-negative, non-spore-forming rods. The strain grows well on ordinary nutrient media (microbiological agar, Hottinger agar). At + 42 ° C, culture growth is maintained. At + 4 ° C, there is no growth. After 24-48 hours of cultivation of the strain on solid nutrient media (microbiological agar of NPO Microgen, Makhachkala), smooth round translucent cream-colored colonies in S-form are formed. According to biochemical properties, the strain is oxide-positive, catalase-positive, forms gelatinase, lysine decarboxylase and ornithine decarboxylase. Does not oxidize glucose, oxidizes fructose, xylose, lactose and maltose. Arginine dehydrolase is absent. Restores nitrates to nitrites. Sensitivity to antibacterial drugs is low: the strain is highly sensitive to fluoroquinolones of the second generation (ciprofloxacin); aminoglycosides (amikacin). The strain was deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" on the basis of the Federal State Budgetary Institution "State Scientific Center for Applied Microbiology and Biotechnology" of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (FBSI SSC PMB) at the address: Russian Federation, 142279, Moscow region, Serpukhov district, pos. Obolensk, under the number No. B-7518 (Certificate of Deposit No. 208 of 12.24.2013).

The strain B. cepacia B-7518 gives a consistently high growth when cultured on solid and liquid nutrient media, while retaining all the properties and is proposed to obtain a somatic antigen used in various diagnostic systems for the determination of antibodies to B. cepacia.

Example 1. Growth characteristics of strain B. cepacia B-7518 when cultured in liquid nutrient media

Three strains were cultured: B. cepacia B-7518 (Volgograd), B. cepacia No. 421 (Moscow) and B. cepacia No. 315 (Rostov-on-Don), obtained from sick children in Russian hospitals Federation. For cultivation, we used nutrient media: microbiological broth (NPO Microgen, Makhachkala) and nutrient broth for the cultivation of microorganisms (FBUN SSC PMB, Obolensk) using a medical shaker S-3.02M. Cultivation conditions: incubation at 37 ° C with stirring at a speed of 20 rpm for 4 hours. The standard seed dose for all samples is 3 billion metric tons / ml (0.4 units of optical density according to McFarland), inoculated into the nutrient medium in a ratio of 1:10. Cultivation for each option was carried out in triplicate. Cultivation control was carried out by metering liquid culture on solid nutrient medium (microbiological agar of NPO Mikrogen, Makhachkala) with the calculation of lg colony forming units in 1 ml (CFU / ml) and the determination of optical density according to McFarland. The results are presented in Table 1.

The data obtained indicate that B. cepacia B-7518 and B. cepacia No. 375 strains are most stable in weight gain on different nutrient media. At the same time, B. cepacia B-7518 gives a culture increase 3 orders of magnitude higher.

B. cepacia B-7518 does not dissociate during cultivation on liquid nutrient media, preserves typical biochemical properties, which, combined with good growth of the culture in liquid nutrient media, makes it the most promising for obtaining somatic antigen.

Example 2. Preparation of antigen

The B. cepacia B-7518 culture was grown on microbiological agar (NPO Mikrogen, Makhachkala) for 24 h at + 37 ° C. The culture was washed off with an isotonic sodium chloride solution (0.85%), pH 7.1 ± 0.1. A suspension of microorganisms was adjusted to a concentration of 1 billion mt / ml in accordance with the turbidity standard (according to McFarland, respectively 0.3 units of optical density). A suspension of microorganisms was inactivated by heating in a water bath (+ 100 ° C) for 15 minutes.

The resulting complex somatic antigen of B. cepacia B-7518 (inactivated) was stored at + 6-8 ° C for a month. Used for formulation of agglutination in vitro, ELISA and immunization of rabbits in order to obtain antiserum (positive control in diagnostic systems).

Example 3. Immunization of rabbits to obtain antisera.

Chinchilla rabbits were immunized with the complex somatic antigen B. cepacia B-7518 in the rear right thigh in the amount of 1.0 ml per 1 animal per 1 injection twice with an interval of 7 days. Then, blood was taken and an antiserum was obtained, which gave an agglutination reaction with the initial antigen in a titer of 1: 8-1: 32. The resulting antiserum was packaged in 0.5 ml in Eppendorf tubes and stored at -20 ° C for 6 months.

The obtained antiserum was used as a positive control in the formulation of the agglutination reaction in test tubes and the ELISA method in order to detect antibodies to burkholderia in biological media.

Antibodies to burkholderia using the obtained antigen and antiserum were determined in a medical immunobiological preparation for the treatment and prevention of acute intestinal infections “Lactoglobulin against opportunistic bacteria and bovine salmonella for oral use” (reg. Certificate No. LSR-002636/08).

Example 4. Formulation of the agglutination reaction to determine antibodies to burkholderii.

Sample preparation of the drug "Lactoglobulin against opportunistic bacteria and Salmonella bovine for oral use."

Vials with lactoglobulin were expanded from metal caps, working dilutions were prepared by two-dimensional titration from 1: 2 to 1:64, using an isotonic sodium chloride solution (pH 7.1 ± 0.1) as a solvent. In prepared agglutination tubes, 2 ml of dilutions of the preparation were added.

Formulation of agglutination reaction:

1. Into test tubes with diluted lactoglobulin samples, 2 ml of the complex somatic antigen of B. cepacia B-7518 (inactivated) was added. As a control, a positive antiserum at a dilution of 1: 8 (positive control of serum) and isotonic sodium chloride solution (negative control of antigen) were used.

2. The contents of the tubes were mixed. The mixture of antigen and lactoglobulin was incubated at 37 ° C for 30 min.

3. The results of the agglutination reaction were taken into account using an agglutinoscope to detect agglutinate sediment in the form of an “umbrella” or “accumulation of granules” in a positive system. The last dilution of the drug with severe agglutination (not less than +++) was considered as diagnostically significant.

The results of the study of the presence of antibodies to burkholderia in 6 series of the drug “Lactoglobulin against opportunistic bacteria and bovine salmonella for oral administration” in the agglutination reaction using the complex somatic antigen of B. cepacia B-7518 are presented in table 2.

Example 5. The formulation of ELISA for the determination of antibodies to burkholderii.

1. Preparation of samples of the drug "Lactoglobulin against opportunistic bacteria and Salmonella bovine for oral use."

Bottles with lactoglobulin were expanded from metal caps, the initial liquid preparation was diluted 1:50. Working dilutions from 1: 100 to 1: 3200 were prepared by two-dimensional titration using an isotonic sodium chloride solution (pH 7.1 ± 0.1) as a solvent.

2. Sensitization of the tablet with antigen.

We used a 96-well polystyrene flat-bottomed plate for the formulation of immunological reactions “Linbro” USA.

Complex somatic antigen B. cepacia B-7518 (inactivated) 100 μl per well in duplicate was added to all wells (according to the number of samples analyzed). The tablet with the antigen was kept at + 4 ° C for 18-24 hours. The unbound components were washed 3 times with phosphate-buffered saline (PBS 7.2 ± 0.2).

Controls:

1) antigen control (negative control)

Antigen was added to the well labeled as antigen control. When test samples were added, PBS was added to the antigen well.

2) control the presence of antibodies (positive control)

From a positive rabbit antiserum, dilutions were prepared: 1:50; 1: 100; 1: 200; 1: 400; 1: 800; 1: 1600. The resulting antiserum dilutions were added to the marked antigen wells. After the study, a calibration curve was built up according to the optical density indices of the positive control antiserum, which was used to determine antibody titers in the test samples.

3) control the absence of antibodies (negative control)

As a negative control for the absence of antibodies, rabbit serum from an unimmunized animal was used, which was diluted with PBS in a ratio of 1:50 and added 100 μl to the corresponding well.

4) conjugate control (positive control)

100 μl of the conjugate working solution, which was developed with the substrate mixture during ELISA, was added to the corresponding well.

3. ELISA

The prepared test samples were introduced at + 37 ° C for 1 h. The unbound components of the reaction were washed with 3 times phosphate-buffered saline (PBS 7.2 ± 0.2).

The commercial preparation Protein A labeled with horseradish peroxidase dry was used as a conjugate (NIIEM Pasteur, St. Petersburg), which was prepared with the addition of bovine serum albumin (BSA) in accordance with the instructions provided by the manufacturers. The BSA conjugate solution was added to the wells with samples and antigen of 100 μl and incubated for 1 h at + 37 ° C. Unbound components of the reaction were washed with 3 times phosphate-buffered saline (PBS 7.2 ± 0.2).

A substrate mixture of OPD (orthophenylenediamine) + hydrogen peroxide of 100 μl was introduced and held for 10-15 minutes at 20 ° C. The reaction was stopped with a Stop reagent (0.1 M sulfuric acid solution) and the result was photographed using an Organon Teknika Reader 530 Version 1.21 micro-scanning device (λ = 492).

4. Accounting for the reaction of ELISA

The antibody titer was determined by the averaged result of the optical density of the sample of the drug, exceeding the optical density in the negative controls by at least 2 times, and the result was compared with calibration data.

The results of the study of the presence of antibodies to burkholderia in 6 series of the drug “Lactoglobulin against opportunistic bacteria and Salmonella bovine for oral administration” using the complex somatic antigen B. cepacia B-7518 by ELISA are presented in Table 3.

Example 6. The use of diagnostic systems for titration of antibodies to burkholderiy in different biological environments.

The agglutination reaction (example No. 4) and the ELISA method (example No. 5) were used to determine antibody titers in different biological samples: lactoserum (the main raw material for lactoglobulin) - 6 samples, rabbit sera - 6 samples, including 3 samples from immunized with B. cepacia B-7518 and 3 samples from non-immunized rabbits. The results are shown in Table 4.

Studies have shown that the agglutination reaction and the ELISA using the somatic antigen of B. cepacia B-7518 give comparable results for the determination of antibody titers in different biological environments - in rabbit sera immunized and non-immunized with B. cepacia B-7518, as well as bovine lactoserum .

Thus, the proposed strain of B. cepacia B-7518 provides a stably high growth when cultured on solid nutrient media while maintaining all the properties, which allows you to use it to obtain somatic antigen for the detection of antibodies to B. cepacia in biological media.

Claims (1)

The strain Burkholderia cepacia B-7518, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" Federal State Budgetary Institution "State Scientific Center for Applied Microbiology and Biotechnology" Rospotrebnadzor under the number B-7518, used to obtain antigen for determination of antibodies to Burkholderia cepacia in bi environments.