RU2566559C1 - Method of differentiation of plague and pseudotuberculosis pathogens as per n-acetyl-beta-d-glucosaminidase activity - Google Patents

Abstract

FIELD: bioengineering.

SUBSTANCE: method of differentiation of plague and pseudotuberculosis pathogens as per N-acetyl-beta-D-glucosaminidase activity provides for production of the suspension of agarinic culture of studied bacteria in concentration (1-5)×109 microcolonies, preparation of synthetic substrate, for this 4-methylumbelliferil-N-acetyl-β-D-glucosaminide is used in amount of 50 mcM. The substrate is dissolved in 2 ml of dimethylformamide. From the produced solution 0.6 ml are taken, and 9.4 ml of 0.1 M phosphate buffer pH 7.4 is added. 20 mcl of prepared solution are mixed with droplet 0.05 ml of suspension of agarinic culture of bacteria in the physical solution in Petri dish. The obtained mixture is incubated for 10-20 minutes at 37°C, then reaction is terminated by addition of 5 mcl of 10 n. alkali solution. Differentiation in UV rays of transilluminator is performed at 366 nm. Bright fluorescence of blue colour is evidence of positive result of the reaction, and confirms that studied strain hydrolyses the prepared substrate and belongs to Yersinia pseudotuberculosis. Absence of incandescence confirms the strain belonging to Yersinia pestis.

EFFECT: invention ensures express diagnostics and differentiation of said bacteria, namely for 10-20 minutes.

3 tbl, 3 ex

Description

The present invention relates to medical microbiology and can be used for rapid diagnosis for all types of strains belonging to the species Yersinia pestis and Yersinia pseudotuberculosis, their differentiation from each other.

Currently, in laboratory diagnostics there is a problem in the differentiation of two closely related and highly homologous (the degree of homology at the DNA level is more than 90%) of Yersinia species of plague and pseudotuberculosis microbes. This circumstance is significant when monitoring the natural foci of plague that actually operate on the territory of the CIS countries.

Pseudotuberculosis microbe strains have a wide geographical distribution, they are pathogenic for a wide range of warm-blooded animals and are often isolated from rodents that live on the territories of natural foci of plague. And although these microorganisms cause different clinical conditions and severity of the disease, many of their phenotypic properties are similar to the plague microbe, which makes their differentiation difficult.

It is known that several chitinases, including exochitinase (N acetyl - β-D-glucosaminidase), are involved in the degradation of naturally-occurring chitin, which is a polymer of N-acetyl-β-D-glucosamine joined by a β-1,4-glycosidic bond which cleaves from the chitin-containing substrates the extreme residues of N-acetylglucosamine easily utilized by the microbial cell (see 1,2).

Damage to the plague pathogen due to deletion of the chiC gene (chitinase, endo-β-N-acetylglucosaminidase), as well as β-hexosaminidase (nghA, YPO2632) due to a shift in the reading frame, which is reflected in the ability of the microbe to block / colonize the flea’s pancreas, is known the formation of aggregates from the pathogen and extracellular matrix, thereby ensuring its transfer to the sensitive host as a result of a bite and subsequent spitting up with excess blood (see 3).

A pseudotuberculosis microbe with the activity of exochitinase enzymes (YTB3365) and β-hexosaminidase (YTB1123) is deprived of this possibility, since the formation of a pancreatic block does not occur in the flea due to destruction of the biofilm by these enzymes. It was not proposed to use the revealed feature to differentiate these pathogens that are close in the genetics but ecologically far from their N-acetyl-β-D-glucosaminidase activity.

Known methods of identification and differential diagnosis, which involve the use of various variants of the polymerase chain reaction (PCR) to detect specific fragments of the genes cafl (plasmid pFra), pla (plasmid pPst) and lcrV (plasmid pCad) of the plague microbe, serological methods (MFA, ELISA, RNat, RNag) for the production of species-specific capsular antigen F1 and other antigens or phenotypic traits (carbohydrate fermentation, urease test, auxotrophy, relation to phages, etc.) (see 4).

However, PCR requires appropriate equipment, specialists, which affects the cost of laboratory tests and the duration of their conduct (at least 2 days).

Closest to the proposed invention is a GenPest test system for detecting Y. pestis DNA by polymerase chain reaction (see 5).

Sodium merthiolate is added to the studied culture samples to a final concentration of 0.01%, followed by heating them at 56 ° C for 30 minutes. After that, 100 μl of the sample was transferred to 1.5 ml microcentrifuge tubes, a lysis solution based on 6 M guanidine thioisocyanate was added, and incubated for 15 min at 65 ° C. Isolation of DNA, PCR is carried out in accordance with the instructions for use of the GenPest test system.

However, the well-known test system requires a lot of time (4-5 hours) and costs for expensive equipment, reagents, a certified laboratory and specialists, which complicates the differentiation method.

The technical task of the invention is to develop a new method that allows in accelerated mode and visual reproduction to differentiate cultures of genetically related Yersinia pestis and Yersinia pseudotuberculosis in vitro.

The problem is achieved in that the method of differentiating pathogens of plague and pseudotuberculosis by N-acetyl-β-D-glucosaminidase activity includes the following stages:

a) obtaining a suspension of agar culture of the studied bacteria in a concentration of (1-5) × 10 ⁹ MK;

b) preparation of a synthetic substrate, for this, a substrate of 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide is taken in an amount of 50 μM, which is dissolved in 2 ml of dimethylformamide,

then 0.6 ml is taken from the resulting solution and 9.4 ml of 0.1 M phosphate buffer pH 7.4 is added to it;

c) mixing the prepared substrate in an amount of 20 μl with a drop of 0.05 ml of a suspension of agar culture of bacteria in physiological solution, placed in a Petri dish;

g) incubation of the mixture at 37 ° C for 10-20 minutes, after which the reaction is stopped by adding 5 μl of 10 N. alkali solution;

d) differentiation in the UV rays of the transilluminator at 366 nm, while bright blue fluorescence indicates a positive result of the reaction, which exhibits N-acetyl-β-D-glucosaminidase activity and confirms that the studied strain hydrolyzes the prepared substrate, therefore, it is identified as strain of Yersinia pseudotuberculosis, and the absence of luminescence confirms its belonging to strains of Yersinia pestis.

The method is as follows.

In order to differentiate, the studied cultures in vitro go through the corresponding stages.

Initially, the studied strains of plague and pseudotuberculosis pathogens were plated separately on Hottinger agar plates with a pH of 7.2 loops to obtain a suspension of bacterial agar cultures at a concentration of (1-5) × 10 ⁹ m.k. Then incubation is carried out for 24-48 hours at 28 ° C.

After that, a synthetic substrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (4-MUF. GlcNAc, Sigma) is prepared. The substrate in an amount of 50 μm is dissolved in 2 ml of dimethylformamide. 0.6 ml was taken from the resulting solution and 9.4 ml of 0.1 M phosphate buffer, pH 7.4, was added to it.

Then, the prepared substrate in an amount of 20 μl is mixed with a drop of 0.05 ml of a suspension of bacterial agar culture in physiological saline placed in a Petri dish.

After this, incubation is carried out for 10-20 minutes at 37 ° C, then the reaction is stopped by the addition of 5 μl of 10 N. solution of any alkali.

Differentiation of the obtained samples is carried out by viewing the samples in the UV rays of the transilluminator at 366 nm.

Thus, the studied strain hydrolyzes the substrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide, exhibiting N-acetyl-β-D-glucosaminidase activity, therefore, it is identified as a strain of Yersinia pseudotuberculosis. If there is no glow, then the fact of the absence of hydrolysis and, accordingly, the absence of N-acetyl-β-D-glucosaminidase activity is confirmed, as a result of which it is concluded that the strain is Yersinia pestis.

Conclusion: N-acetyl-β-D-glucosaminidase activity of Yersinia pestis and Yersinia pseudotuberculosis strains is considered as a differential trait.

Example 1

As the studied strains, 14 cultures of Y. pseudotubeculosis of different serotypes and from different sources of excretion were used. All of them had N-acetylglucosaminidase activity (see table 1). The stages of the method described above. As a substrate used 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (4-MUF. GlcNAc, Sigma) in an amount of 20 μl.

The calculation of the results is shown in table 1, which shows that N-acetyl-β-D-glucosaminidase activity is found in all

crop research. Therefore, they belong to the species Yersinia pseudotuberculosis.

Example 2

For study, 22 museum strains (collection of the Rostov-on-Don antiplague institute) of another representative of the Yersinia genus, Y. enterocolitica, were taken, which also effectively cleaved the proposed substrate (see table 2). The technology of the method is the same as in example 1. All strains had N-acetylglucosaminidase activity. As a substrate, 20 μl of 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide was used.

From table 2 it is seen that N-acetyl-β-D-glucosaminidase activity is found in all cultures taken in the study. Thus, it is possible to differentiate the studied strains as non Yersinia pestis.

Example 3

We studied a collection of 50 strains of the plague microbe of the main and minor subspecies stored in the lyophilized state in the Museum of Living Cultures of the Russian Orthodox Church. Strains were isolated in different natural foci of the plague of the former USSR and the world, in different years and from different carriers. The technological stages of the process are the same as in example 1.2.

As a result, none of the strains was able to hydrolyze the synthetic substrate 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (4-MUF. GlcNAc, Sigma) in an amount of 20 μl (see table 3). From tables 1,2 it is seen that in all tested strains of the causative agent of pseudotuberculosis and Y. enterocolitica, regardless of serotype, place, time and source of excretion, N-acetyl-β-D-glucosaminidase activity is detected, while 50 strains of the plague microbe from different natural foci were deprived of the ability to cleave a synthetic substrate.

Conclusion: it is possible to differentiate according to the studied attribute.

The use of the present invention allows to carry out in an accelerated mode, namely within 10-20 minutes, and clearly reproduce the differentiation of Yersinia pestis from Yersinia pseudotuberculosis in vitro, which allows this method to be used as an express diagnosis in an epidemic situation and monitoring of large areas, since gives time savings, which is of great importance for making urgent sanitary and epidemic decisions.

Information sources

1. Enzyme nomenclature -1984, p. 313.

2. The nomenclature of enzymes. Moscow. 1979, p. 148.

3. Erickson D.L., Jarrett C.O., Callison J.A., Fischer E.R., Hinnebusch B.J. Loss of a biofilm-inhibiting glycosyl hydrolase during the emergence of Yersinia pestis. J. Bacteriol., 2008, v. 190, N 24, p. 8163-8170.

4. Brubaker R.R. The recent emergence of plague: a process of felonious evolution. Microbial Evolution, 2004, v. 47, p. 293-299.

5. Laboratory diagnosis of dangerous infectious diseases. Practical Guide / Ed. academician of RAMS, prof. G.G. Onishchenko, Corr. RAMS prof. V.V. Kutyreva. - M .: OJSC “Publishing house“ Medicine ”, publishing house“ Shiko ”, 2009, p. 21-61.

Claims (1)

A method for differentiating pathogens of plague and pseudotuberculosis according to N-acetyl-β-D-glucosaminidase activity, comprising the following stages:
a) obtaining a suspension of agar culture of the studied bacteria in a concentration of (1-5) × 10 ⁹ MK;
b) preparation of a synthetic substrate; to do this, take in its quality a substrate of 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide in an amount of 50 μM, which is dissolved in 2 ml of dimethylformamide, then 0.6 ml is taken from the resulting solution and 9.4 ml of 0 is added to it , 1 M phosphate buffer pH 7.4;
c) mixing the prepared substrate in an amount of 20 μl with a drop of 0.05 ml of a suspension of agar culture of bacteria in physiological solution, placed in a Petri dish;
g) incubation of the mixture at 37 ° C for 10-20 minutes, after which the reaction is stopped by adding 5 μl of 10 N. alkali solution;
d) differentiation in the UV rays of the transilluminator at 366 nm, while bright blue fluorescence indicates a positive result of the reaction, which exhibits N-acetyl-β-D-glucosaminidase activity and confirms that the studied strain hydrolyzes the prepared substrate, therefore, it is identified as strain of Yersinia pseudotuberculosis, and the absence of luminescence confirms its belonging to strains of Yersinia pestis.