RU2288274C1 - Method for assay of sugar inhibitory capacity degree on adhesion process of cholera vibrio - Google Patents

Abstract

FIELD: medicinal microbiology.

SUBSTANCE: adhesion of cholera vibrio is carried out in the presence of carbohydrates as inhibitors that interact with lectin receptors of cholera vibrio and inhibit their adhesive properties. Method involves preparing 1% carbohydrate solution in 1 ml of 1 billion part of cholera vibrio suspension that are incubated at temperature 37°C for 30-40 min in wet chamber followed by preparing preparation for microscopic investigation. The degree of inhibitory capacity of sugars on adhesion process in the analyzed culture of cholera vibrio is determined by the adhesion inhibition scale: weak inhibition index, in limits 0.36-0.9; middle inhibition index, 0.176-0.35, and strong inhibition index, 0.01-0.175. Using the proposed method provides enhancing biotechnological possibilities of assay. Invention can be used for characterization of pathogenicity of cholera vibrio.

EFFECT: improved assay method.

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Description

The present invention relates to medical microbiology and can be used to characterize the pathogenicity of cholera vibrios.

Adhesion of bacterial cells to cells of macroorganism tissue remains poorly understood. Microbial cells of both normal and pathogenic microflora attach to the surface of the mucous membrane through rather complex mechanisms. The path to understanding these mechanisms lies through the simultaneous study of adhesion organelles in bacteria and surface structures of macroorganism tissues, in relation to which adhesion is carried out.

The chemical nature of cholera adhesins, as well as adhesins of other vibrios, continues to be poorly studied. There is little information that adhesins are protein in nature and have pectin properties due to the carbohydrate-binding domain, which is expressed in hemagglutination.

A known method for determining the adhesive ability of cholera vibrios (see AS USSR No. 1306107, class C 12 N 1/00, C 12 Q 1/02, B 24, 1990), which consists in the fact that the test culture is incubated with 0 , 5-1% suspension of rabbit erythrocytes used as a substrate and taken in equal volumes for 25-40 minutes, then by preparation of microscopic preparations, the adhesive ability is determined by the number of vibrios attached to the substrate.

However, the known method allows to detect the fact of the adhesive ability of the cholera vibrios of the studied culture and does not make it possible to determine the presence or absence of carbohydrate-binding domains or pectin receptors involved in the adhesion process, and also to evaluate the effect of various substances, namely sugars on the adhesion process.

For the prototype, a method was chosen for evaluating the adhesive properties of enteropathogenic bacteria on red blood cells (see V.I. Brillis, article "Methods for Studying the Adhesive Process of Microorganisms", Journal of Laboratory, No. 4, Moscow, Medicine, 1986). On a glass slide, 1 drop of 1 billion suspension of cholera vibrios and 1 drop of 5-10% suspension of red blood cells prepared in 0.1 M Na phosphate solution are mixed, incubated at 37 ° C for 30 minutes in a humid chamber, after which the smears are fixed with alcohol and stained according to Romanovsky-Giemsa, while the adhesion study is carried out under a light microscope, followed by an average adhesion index (SPA), the count is carried out on 100 red blood cells, and the adhesion of cholera vibrios is considered zero at SPA from 0 to 1; average from 1.01 to 3; high - above 3.

The disadvantage of the prototype is that in the presence of adhesion it is impossible to study the specificity of the receptors of the cholera vibrios themselves involved in adhesion, the nature of the targets for adhesion on the surface of red blood cells, and the effect of various substances, in particular sugars, on the adhesion process. These circumstances reduce the biotechnological capabilities of the prototype and narrow its use for the diagnosis and treatment of cholera.

The objective of the invention was to increase the biotechnological capabilities of the method in determining the inhibitory ability of sugars on the adhesion of cholera vibrios.

The problem is achieved in that in the known method for determining the inhibitory ability of sugars to adhesion of cholera vibrios, including incubating the test culture with red blood cells, adhesion of cholera vibrios is carried out in the presence of an inhibitor, which is used as carbohydrates, the latter interact with pectin receptors of cholera vibrios and inhibit their adhesive properties, to do this, prepare a 1% solution of carbohydrate in 1 ml of 1 billion suspension of cholera vibrios, which are incubated in a thermostat at at a temperature of 37 ° C for 30-40 minutes, then rabbit erythrocytes and a suspension of vibrios with carbohydrates are applied in equal volumes to the glass, incubated at a temperature of 37 ° C for 30-40 minutes, followed by the preparation of microscopic preparations and determined on a scale degree of sugar inhibition of adhesion of the test culture. At the same time, on the adhesion inhibition index (PIA) scale, it is determined: a weak inhibition index in the range of 0.36-0.9, an average of 0.176-0.35 and a high of 0.01-0.175.

The method is as follows.

As inhibitors, 1% solutions of various carbohydrates are used, for example, glucose, mannose, fructose, galactose or carbohydrate-containing preparations.

To carry out the method, red blood cells of a rabbit, a ram or a human can be used.

Initially, 1 billion suspensions of cholera vibrios of the studied culture are prepared. Then a dry sample of carbohydrate in an amount of 10 mg / ml (1% solution) is combined with a suspension of cholera vibrios and incubated at a temperature of 37 ° C for 30-40 minutes.

Then, 1 drop of 5% erythrocytes (human) and 1 drop of a suspension of vibrios with carbohydrates are applied to a glass slide, incubated at a temperature of 37 ° C for 30-40 minutes in a moist chamber. After this, a smear is prepared, fixed with a mixture of Nikiforov and stained according to the method of V.I. Brillis.

Accounting is carried out through a microscope in two stages:

Stage 1 - consider the average adhesion index (SPA) according to the method of V.I. Brillis. Counting is carried out on 100 red blood cells. For example, if 200 vibrios attached to 100 red blood cells, then SPA = (200: 100) = 2. From table 1 it is seen that this indicator corresponds to the average adhesiveness.

Stage II - assess the average adhesion inhibition index (PIA) on a scale (see table 1).

The proposed scale is based on the examples performed with different inhibitors, for which different carbohydrates were taken, and the following dependence was determined by their interaction with pectin receptors of cholera vibrios of various strains. The calculation was based on the average number of vibrios attached to one red blood cell using a carbohydrate. For example, if 25 vibrios attached to 100 red blood cells, then PIA = 25: 100 = 0.25. PIA in the range of 0.36-0.9 is a weak indicator of inhibition, an average of 0.176-0.35 and a high of 0.01-0.175.

In each experiment, two indicators of SPA (without carbohydrate) and PIA (reaction with carbohydrates) are compared. If, for example, SPA = 2, and PIA = 0.25, then we conclude that the carbohydrate effectively inhibited adhesion (within the average).

Thus, there is an average and high rate of adhesion inhibition in comparison with the adhesion control according to the method of V.I. Brillis. We conclude about the presence and specificity of pectin receptors involved in adhesion, as well as the effect of a certain sugar on this process.

Example 1

Determine the inhibitory ability of carbohydrate on the adhesion of cholera vibrios in strain V. Cholerae cholerae 569 B.

As an inhibitor, galactose in forms D and β and glucose are taken. Prepare a 1% solution of galactose and glucose in 1 ml of 1 billion suspension of cholera vibrios and incubate at a temperature of 37 ° C for 30-40 minutes. Then, 1 drop of 5% sheep erythrocytes and 1 drop of a suspension of vibrios with galactose and glucose are applied to a glass slide. Incubated at a temperature of 37 ° C for 30-40 minutes in a humid chamber, prepare a smear, fix with a mixture of Nikiforov and stain according to Romanovsky-Giemsa. After the incubation time has elapsed, an adhesion inhibition indicator is considered under a light microscope - the average number of vibrios attached to one erythrocyte when using galactose in the forms D and β and glucose. In parallel, they put the adhesion control (in full accordance with the method of V.I. Brilis), then compare two indicators: the average adhesion inhibition index (PIA) and the average adhesion index (SPA) (see table 2).

From table 2 it is seen that the strain is highly adhesive, SPA = 5. Galactose in forms D and P reduces adhesion to 1.7 (PIA), however, according to table 1, when SPA = 1.01-2.9, the strains are considered medium adhesive. Thus, we conclude that the galactose-specific receptor (pectin) does not dominate during adhesion, in contrast to glucose-specific pectin, which is actively involved in adhesion, since glucose inhibits adhesion to PIA = 0.1, which according to table 1 corresponds to the average adhesion inhibition.

Example 2

The strains V. Cholerae O 139 (Bengal) were used as the studied cultures, and β-galactose and N-acetylmannosamine as inhibitors.

The sequence of steps of the method is the same as in example 1, and their results are summarized in table 3.

Analyzing the indicators of Table 3, we conclude that the Bengal strains have moderate adhesion, taking into account the SPA, according to Table 1, a galactose-specific receptor takes place and is actively involved in adhesion, since PIA falls within the range of a high adhesion inhibition rate (0.01- 0.175), the glucose-specific pectin receptor is less pronounced - PIA corresponds to the average adhesion inhibition index (0.176-0.35) and generally there is no pectin recognizing N-acetylmannosamine, since there are more than 1 in all six PIA strains.

Example 3

It differs from Example 1 and 2 in that the strains V. Cholerae cholerae 569 B, V. Cholerae Eltor 1310, 5879, 18210, 3119, V. Cholerae O 139 16064, 16065, 16131, 16063, 16077 are used as the cultures under study. glucosalan, a rehydration therapy drug containing glucose, is used as an adhesion inhibitor.

The results of the example are summarized in table 4.

Thus, according to the results of the study, it was found that glucosalan effectively inhibits the adhesion of cholera vibrios of various strains.

Using the present invention, due to carbohydrate-containing inhibitors, it is possible to detect the specificity of the carbohydrate-binding domain in the cholera vibrios of the studied culture for sugars, which helps to reduce their adhesive ability due to the binding of individual pectin receptors of cholera vibrios with a specific carbohydrate.

At the same time, on the scale of adhesion inhibition indicators (PIA) developed by specialists of the Rostov-on-Don research anti-plague institute, it is possible to quickly determine the degree of sugar inhibition of the studied culture, which expands diagnostic capabilities in biotechnology and can be used in practical use in the treatment of cholera and its prevention in epidemic outbreaks, since adhesion is the initial stage in the pathogenesis of this infectious disease.

Table 1 Estimation of average adhesion carbohydrate adhesion inhibition assessment SPA = 0-1 - not adhesive PIA = 0.36-0.9 weak indicator of inhibition of adhesion SPA = 1.01-2.9 - adhesive (medium adhesiveness) PIA = 0.176-0.35 average adhesion inhibition SPA> 3 - high adhesion PIA = 0.01-0.175 high adhesion inhibition rate

table 2 Strain number SPA (control) Adhesion Inhibition Rate (PIA) D-galactose β-galactose glucose V. Cholerae cholerae 569 B 5.0 ± 0.2 1.5 ± 0.03 1.7 ± 0.05 0.1 ± 0.02

Table 3 Strain number Average adhesion index (SPA) adhesion inhibition index (PIA) D-galactose Glucose N-acetylmannosamine V. Cholerae O 139 16064 1.8 0.08 ± 0.02 0.25 ± 0.03 1.5 ± 0.02 16065 2.0 0,07 ± 0,1 0.28 ± 0.04 1.8 ± 0.04 16131 1,56 0.05 ± 0.03 0.47 ± 0.05 1.5 ± 0.03 16063 2.0 0.03 ± 0.01 0.35 ± 0.02 1.5 ± 0.03 16077 2,3 0.06 ± 0.05 0.5 ± 0.1 1.9 ± 0.01

Table 4 Strain number SPA (control) glucosalan adhesion inhibition index (PIA) V. Cholerae cholerae 569 B 5.0 ± 0.2 0.2 ± 0.03 V. Cholerae Eltor 1310 4,5 ± 0,1 0.2 ± 0.03 5879 3.5 ± 0.02 0.1 ± 0.02 18210 3 ± 0.2 0.2 ± 0.02 3119 3.5 ± 0.09 0.2 ± 0.02 16064 1.8 ± 0.03 0.25 ± 0.03 16065 2 ± 0.09 0.27 ± 0.03 16131 1.5 ± 0.1 0.38 ± 0.05 16063 2 ± 0.05 0.25 ± 0.03 16077 2.3 ± 0.02 0.25 ± 0.02

Claims (1)

A method for determining the degree of inhibitory ability of sugars on the process of adhesion of cholera vibrios, including incubation of the test culture together with red blood cells, characterized in that the adhesion of cholera vibrios is produced in the presence of an inhibitor, which is used as carbohydrates, the latter interact with pectin receptors of cholera vibrios and inhibit their adhesive properties for this, prepare a 1% solution of carbohydrate in 1 ml of 1 billion suspension of cholera vibrios, which are incubated at a temperature of 37 ° C for 30-40 minutes in a wet chamber with subsequent preparation of microscopic preparations in which the degree of adhesion inhibition is determined on the scale of adhesion inhibition indicators (PIA), while for a weak degree of adhesion inhibition, PIA is taken in the range 0.36-0.9, the average degree in the range 0.176- 0.35 and high - 0.01-0.175.