RU2465318C1 - Yersinia pseudotuberculosis test strain for differentiation of yersinia pseudotuberculosis bacteria genetic group iia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Yersinia pseudotuberculosis 563 bacterial strain is recovered from a patient suffering pseudotuberculosis, deposited in the collection of the Russian Science and Research Antiplague Institute Microbe, No. KM 212 and is applicable as a test strain for differentiation of Yersinia pseudotuberculosis bacteria genetic group II_A. The characteristic of the strain is the content of chromosomal gene of the Y. pseudotuberculosis YPMa/YPMc (urtA/C) superantigen determined by PCR method.

EFFECT: invention shall enable epidemiologic study of sporadic and group cases of pseudotuberculosis: specifying an infection source in a hotbed of the disease and identifying transmission factors, as well as monitoring strain circulation at various territories.

2 dwg, 1 tbl

Description

The invention relates to medical microbiology and is an original strain of the pseudotuberculosis pathogen containing the Y. pseudotuberculosis YPMa / YPMc chromosome gene (ypmA / C) and used as a test strain for the differentiation of Y.pseudotuberculosis bacteria of genetic group IIa.

Pseudotuberculosis is registered almost everywhere in Russia, while the highest incidence rates are characteristic of temperate and cold climates (Siberian, Far Eastern, North-Western Federal Districts).

Pseudotuberculosis in various geographical regions is known to have certain clinical symptoms (Fukushima N., Matsuda Y., Seki R., et al. Geographical Heterogeneity between Far Eastern and Western Countries in Prevalence of the Virulence Plasmid, the Superantigen Yersinia pseudotuberculosis-Derived. Mitogen, and the High-Pathogenicity Island among Yersinia pseudotuberculosis Strains. J. Clin. Microbiol. 2001; 39 (10): 3541-3547): In Europe, this infection is mainly characterized by the presence of fever, gastrointestinal symptoms and mesenteric lymphadenitis, whereas in Russia these are systemic manifestations such as rash, erythema nodosum, arthritis.

This polymorphism of the clinical manifestations of the disease is due to the presence of a large set of pathogenicity factors of chromosomal and plasmid nature in the pathogen. Thus, the genes of the “island” of high pathogenicity of HPI located on the chromosome are responsible for the biosynthesis and regulation of the iron utilization system necessary for the expression of the highly virulent phenotype of enterobacteria (Carniel E. The Yersinia high-pathogenicity island: an iron-uptake island. Microbes and Infection, 3, 2001, 561-569). Superantigens of YPMs in the host organism stimulate the polyclonal activation of T-lymphocytes (CD4 +, CD8 +). In this case, from 10 to 40% of all lymphocytes are involved in the interaction. This leads to overproduction of cytokines, such as IL-2, TNF-α, IFN-γ, which can cause toxic shock syndrome (Shurygina I.A., 2003). Three groups of superantigens are known - YPMa, YPMb, YPMc encoded by the corresponding ypmA, ypmB and ypmC genes (Carnoy C., Floquet S., Marceau M., et al. The superantigen gene ypm is located in an unstable chromosomal locus of Yersinia pseudotuberculosis. J. Bacteriol. 2002, 184 (16): 4489-99). YAPI is an adhesive “island” of Yersinia pathogenicity; it carries genes encoding type IV drank (filamentous structures extending from the surface of a bacterial cell, shorter and more numerous than flagella). Type IV dranks are involved in a variety of bacterial functions, including adhesion to Peyer's plaque cells, bacteriophage adsorption, plasmid transfer and flagellin independent motility (Collyn F., Léty MA, Nair S., et al. Yersinia pseudotuberculosis harbors a type IV pilus gene cluster that contributes to pathogenicity. Infect Immun., 2002, 70 (11): 6196-205). Plasmid with a pier. weighing 82 MDa (pVM82), present in some strains of Y. pseudotuberculosis, has an immunosuppressive and antifagocytic effect on the immune structures of a macroorganism (Dubrovina V.I., Golubinsky E.P., Borsuk G.I. and author Features of phagocytosis Yersinia pseudotuberculosis with a different set of plasmids. Medical parasitol., 1999, 4: 50-53). M.V. Chesnokova et al. (Chesnokova M.V., Klimov V.T., Maramovich A.S. Genotyping of Yersinia pseudotuberculosis isolated in Siberia and the Far East. Zh. Microbiol., 2006, 6, 20-25) showed that strains of Y.pseudotuberculosis possessing the plasmid pVM82 cause a more severe course of pseudotuberculosis.

Previously, Y. pseudotuberculosis strains were examined by the method of NDP for the presence of such virulence genes as superantigen genes (ypmA, ypmB and ypmC), genes of the “island” of high pathogenicity HPI, and were divided into genetic groups (Fukushima H., Matsuda Y. , Seki R., et al. Geographical Heterogeneity between Far Eastern and Western Countries in Prevalence of the Virulence Plasmid, the Superantigen Yersinia pseudotuberculosis-Deried Mitogen, and the High-Pathogenicity Island among Yersinia pseudotuberculosis Strains. J. Clin. Microbiol. 2001; 39 (10): 3541-3547). The disadvantage of this study is that it did not take into account other pathogenicity factors - the adhesive “island” of pathogenicity of yersinia and plasmid pVM82.

Currently, for the epidemiological investigation of cases of sporadic and group diseases of pseudotuberculosis and for monitoring pathogens in various territories, the content of Y. pseudotuberculosis plasmids is studied, which are isolated by T. Kieser in several stages, and the presence of genes of the “island” of high pathogenicity is determined by PCR HPI and superantigen (Chesnokova M.V., Klimov V.T., Maramovich A.S. Genotyping of Yersinia pseudotuberculosis isolated in Siberia and the Far East. - Zh. Microbiol., 2006, 6, 20-25). The analogue strain could not be obtained.

For the prototype, a strain of Y. pseudotuberculosis KM-9/2160 was selected (Messorosh V.G. Biological properties of Yersinia pseudotuberculosis and improvement of laboratory diagnosis of pseudotuberculosis. Author. Diss. ... Ph.D. Medical Sciences. - St. Petersburg, 1993, 18 pp.), used as a standard for determining virulence, deposited in the collection of pathogenic microorganisms of RosNIPCHI "Microbe". The degree of virulence was determined by conjunctival infection of guinea pigs, enteric infection of white mice and guinea pigs, and infection of tissue cultures of the HEP-2, HeLa lines. A study of the presence of virulence genes has not been conducted. Therefore, the use of the strain to perform monitoring tasks for the circulation of the causative agent of pseudotuberculosis and the search for the source of infection is impractical.

The invention is directed to obtaining a test strain of Y. pseudotuberculosis genetic group IIa.

EFFECT: differentiation of bacteria Yersinia pseudotuberculosis of genetic group IIa by the content of virulence genes, which will make it possible to establish the source of infection in the outbreak and identify transmission factors, as well as monitor the circulation of strains in different territories.

The obtained strain Y. pseudotuberculosis 563 was deposited in the collection of pathogenic microorganisms of RosNIPCHI “Microbe” KM 212. The strain is characterized by the content of the chromosomal gene of the superantigen Y.pseudotuberculosis YPMa / YPMc (ypmA / C).

A strain of the family Enterobacteriaceae genus Yersinia species Y. pseudotuberculosis, isolated from a patient with pseudotuberculosis of exanthema form of mild severity, Zima, 1999

Morphological and physiological characteristics. Gram-negative asporogenous capsuleless rod-shaped cells 1-3 microns, weakly mobile at temperatures below 30 ° C, at 37 ° C they are motionless. On liquid nutrient media under aerobic conditions at 28 ° C or at 37 ° C form a uniform turbidity. On Hottinger agar at 28 ° C, after 48 h, convex transparent grayish-yellow shiny colonies with a relatively even edge and a raised center up to 1.2-1.8 mm in size are formed. On a medium with bromothymol blue at 28 ° С, after 48 h, on a blue-blue background, the medium forms small (1-2 mm), bluish-green, with a raised center, convex, dry, with a matte bloom of a colony with a scalloped edge.

Biochemical activity. It ferments with the formation of acid: glucose, mannitol, xylose, does not ferment lactose, sucrose, sorbitol. Samples for indole and hydrogen sulfide are negative. It produces catalase, does not produce oxidase. Hydrolyzes urea, does not ferment salicin. Does not produce ornithine decarboxylase.

Antigenic properties. Enters into agglutination reaction with diagnostic serum to Y. pseudotuberculosis O: 1.

Genetic features. Contains the YPMa / YPMc superantigen chromosomal gene (ypmA / C); there is no gene for the adhesive island of pathogenicity of Yersinia YAPI (pilPQ), a chromosome gene for the island of high pathogenicity HPI (fyuA) and the gene for superantigen Yersinia YPMb (ypmB), as well as plasmid pVM82 (dotO).

Compared to the analogue strain, the presence of a chromosomal pathogenicity factor, YAPI, is also studied. The advantage of this study is to use only the PCR method to search for all virulence genes.

The invention is illustrated by drawings.

Figure 1 presents the results of PCR with strain Yersinia pseudotuberculosis No. 563 (group IIa), where M is a molecular weight marker for DNA (10000 b.p .; Eurogentec); 1 - ypmA / C (422 b.p.); 2 - ypmB (268 b.p.); 3 - fyuA (149 b.p.); 4 - pilPQ (569 b.p.); 5 - dotO (2811 b.p.).

Figure 2 presents the results of PCR with strains of Yersinia pseudotuberculosis No. 563 lanes (1-5), No. 540 lanes (6-10) and No. 608 lanes (11-15), where M is the molecular weight marker of DNA (VI .; Eurogentec ); 1, 6, 11 - ypmA / C (422 b.p.); 2, 7, 12 - ypmB (268 b.p.); 3, 8, 13 - fyuA (149 b.p.); 4, 9, 14 - pilPQ (569 b.p.); 5, 10, 15 - dotO (2811 b.p.).

The invention is implemented as follows.

Example 1. A method of obtaining a strain

A pure strain culture is grown on Hottinger agar at 28 ° C. for 24 hours.

A suspension of bacteria is prepared with a concentration of 10 ⁸ mk / ml in sterile distilled water, 100 μl of the suspension is boiled for 5 minutes, centrifuged at 10 thousand rpm / min for 1 minute, and a supernatant is used. Bacteria are examined by polymerase chain reaction with primers for gene regions encoding pathogenicity factors Y. pseudotuberculosis manufactured by Syntol LLC (Moscow) (Table 1)

The reaction mixture for the NDP is prepared using standard Fermentas reagents according to the attached instructions: 2.5 μl 10x Taq buffer with (NH ₄ ) ₂ SO ₄ , 2 mM / μl MgCl ₂ , 0.2 mm / μl of each dNTP, 0.4 μM / μl of forward and reverse primers, 0.625 U Taq-DNA polymerase, with the addition of 4 μl of the test sample to a final volume of 25 μl. PCR is carried out according to the program: 1 cycle of 95 ° C - 5 min; 25 cycles 94 ° С - 1 min, 55 ° С - 1 min, 72 ° С - 1 min; 1 cycle 72 ° C - 7 min. To take into account the results of PCR, electrophoresis was performed on a 1.5% agarose gel at a constant voltage of 75 V in TBE buffer (0.9 M Tris, 0.9 M boric acid, 20 mM EDTA-Naz) stained with ethidium bromide.

Figure 1 presents the result of electrophoretic separation of DNA fragments obtained after the NDP.

It is seen that strain 563 has one DNA fragment, 422 bp in length. (lane 1), which corresponds to the plot of the Y strains of the ypmA / C gene. There are no amplicons on tracks 2, 3, 4, 5.

Example 2. The study of DNA strains of Yersiniapseudotuberculosis.

Using the NDP method using primers for the Yersinia chromosomal genes (fyuA, pilPQ, ypmA / C, ypmB) and the plasmid gene (dotO), DNA strains that were isolated during the investigation of the pseudotuberculosis outbreak in Zima were studied. Strain No. 540 isolated from flushing with cabbage; No. 608 isolated from rat. As a control, the strain Y. pseudotuberculosis KM 212 was used. The results of the study are presented in figure 2.

It is seen that in all the studied strains of Y. pseudotuberculosis, one DNA fragment with a length of 422 bp is present. (lanes 1, 6, 11), like the obtained test strain and, accordingly, these strains can be assigned to genetic group IIa. Thus, the fact of genetic similarity of the studied outbreaks was established, which allows us to confirm the commonality of their origin and to establish the source and transmission factor of the infection.

Thus, the claimed strain is suitable for differentiation of bacteria Yersinia pseudotuberculosis of genetic group IIa by the content of virulence genes, which will allow to establish the source of infection in the focus of the disease and identify transmission factors, as well as monitor the circulation of the strains in different territories.

Claims (1)

Test strain Yersinia pseudotuberculosis, RosNIPCHI "Microbe" KM 212, for differentiation of bacteria Yersinia pseudotuberculosis of genetic group II _A.

Images