RU2566554C1 - Stenotrophomonas maltophilia STRAIN USED FOR OBTAINING OF ANTIGEN FOR IDENTIFICATION OF ANTIBODIES TO Stenotrophomonas maltophilia IN BIOLOGICAL MEDIUMS - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: Stenotrophomonas maltophilia strain is deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the registration number V-7520.

EFFECT: strain can be used for obtaining of anti-gene for identification of antibodies to Stenotrophomonas maltophilia in biological environments.

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Description

The invention relates to medical microbiology and biotechnology, and relates to a strain of Stenotrophomonas maltophilia, which can be used to obtain an antigen for determining antibodies to Stenotrophomonas maltophilia in biological media.

The incidence of infections associated with S. maltophilia in the resuscitation and intensive care units has increased markedly. The choice of antibacterial drugs for the management of such infections is a big problem for the microbiological laboratory and clinicians. 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 Stenotrophomonas maltophilia in biological media on an industrial scale, a standard strain of Stenotrophomonas maltophilia is required for its growth, biochemical and antigenic characteristics, which will ensure biomass growth while maintaining properties.

The aim of the invention is the selection of a strain of Stenotrophomonas maltophilia, 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 Stenotrophomonas maltophilia B-7520 has morphological, tinctorial, biochemical and cultural characteristics typical of S. maltophilia. Cells are small gram-negative motile rods. On the MPA medium (NPO Microgen, Makhachkala), Stenotrophomonas maltophilia B-7520 grows in the form of translucent colonies (diameter 1-2 mm). Stenotrophomonas maltophilia B-7520 according to biochemical properties: oxide-positive, catalase-positive, form gelatinase, form lysine decarboxylase. Arginine dehydrolase and ornithine decarboxylase are absent. Oxidize glucose, do not oxidize fructose, xylose, lactose and maltose, and reduce nitrates to nitrites. The virulence for laboratory animals in the LD50 assay on white mice was 8 billion mt / ml. The sensitivity to antibacterial drugs is low: a highly sensitive culture 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" Rospotrebnadzor, reg. No. В-7520 (Certificate of Deposit No. 210 dated 12.24.2013).

The strain Stenotrophomonas maltophilia B-7520 gives a consistently high growth when cultured on solid and liquid nutrient media, while preserving all the properties and is proposed to obtain a somatic antigen used in various diagnostic systems for the determination of antibodies to S. maltophilia.

Example 1. Growth characteristics of the strain Stenotrophomonas maltophilia B-7520 when cultured in liquid nutrient media

Two strains were cultured: Stenotrophomonas maltophilia B-7520 and Stenotrophomonas maltophilia 377 (Rostov-on-Don), obtained from sick children of the pulmonology department in the bacteriological laboratory of the Children's Regional Hospital. For cultivation, nutrient media were used: microbiological broth (NPO Microgen, Makhachkala) and nutrient broth for microorganism cultivation (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 inoculum dose for all samples, 3 billion mt / ml (0.4 units of optical density according to Mac-Farland), was 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 a solid nutrient medium (microbiological agar of NPO Microgen, 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 the strain Stenotrophomonas maltophilia B-7520 was the most stable in mass gain on different nutrient media. At the same time, Stenotrophomonas maltophilia B-7520 gave a culture increase of 2.5-3 orders of magnitude higher.

Stenotrophomonas maltophilia B-7520 did not dissociate during cultivation on liquid nutrient media and retained typical biochemical properties, which, combined with a good growth of the culture in liquid nutrient media, makes it the most promising for obtaining somatic antigen.

Example 2. Preparation of antigen

The culture of Stenotrophomonas maltophilia B-7520 was grown on microbiological agar (NPO Mikrogen, Makhachkala) for 24 hours 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 obtained complex somatic antigen Stenotrophomonas maltophilia B-7520 (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 antiserum

Chinchilla rabbits were immunized with the complex somatic antigen Stenotrophomonas maltophilia B-7520 in the rear right thigh in the amount of 1.0 ml per animal for 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 resulting antiserum was used as a positive control in the formulation of the agglutination reaction in vitro and the ELISA to detect antibodies to Stenotrophomonas maltophilia in biological media.

Antibodies to Stenotrophomonas maltophilia 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 stenotrophomonas

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 the agglutination reaction

1. Into test tubes with diluted lactoglobulin samples, 2 ml of the complex somatic antigen Stenotrophomonas maltophilia B-7520 (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 Stenotrophomonas maltophilia in 6 series of the drug “Lactoglobulin against opportunistic bacteria and Salmonella bovine for oral administration” in the agglutination reaction using the complex somatic antigen Stenotrophomonas maltophilia B-7520 are presented in table 2.

Example 5. Formulation of ELISA for the determination of antibodies to Stenotrophomonas maltophilia

The preparatory phase of the IFA

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 original 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. Tablet sensitization by antigen

A 96-well polystyrene flat-bottomed plate was used for immunological reactions "Linbro" USA.

In all wells (in accordance with the number of analyzed samples), the complex somatic antigen Stenotrophomonas maltophilia B-7520 (inactivated) was added at 100 μl per well in duplicate. 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).

3. 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.

4. ELISA

The prepared test samples were added at + 37 ° C for 1 hour. 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 (Pasteur Research Institute of Nuclear Medicine, St. Petersburg), which was prepared with the addition of bovine serum albumin (BSA) in accordance with the instructions provided by the manufacturers, was used as a conjugate. The BSA conjugate solution was added to the wells with samples and antigen of 100 μl and incubated for 1 hour 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 RPD + hydrogen peroxide of 100 μl was added and kept 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).

5. 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 compared the result with calibration data.

The results of the study of the presence of antibodies to Stenotrophomonas maltophilia in 6 series of the drug "Lactoglobulin against opportunistic bacteria and Salmonella bovine for oral administration" using the complex somatic antigen Stenotrophomonas maltophilia B-7520 by ELISA are presented in Table 3.

Example 6. The use of diagnostic systems for titration of antibodies to Stenotrophomonas maltophilia 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 Stenotrophomonas maltophilia B-7520 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 Stenotrophomonas maltophilia B-7520 give comparable results for the determination of antibody titers in different biological media - in rabbit sera, immunized and non-immunized Stenotrophomonas maltophilia B-7520, as well as bovine serum.

Thus, the proposed strain of S. maltophilia B-7520 provides a stably high growth when cultured on solid nutrient media while maintaining all the properties, which allows it to be used to obtain a somatic antigen for determining antibodies to S. maltophilia in biological media.

Claims (1)

The strain Stenotrophomonas maltophilia, deposited in the State collection of pathogenic microorganisms and cell cultures "GKPM-Obolensk" under the number B-7520, used to obtain antigen for the detection of antibodies to S. maltophilia in biological environments.