RU2465319C1 - Yersinia pseudotuberculosis test strain for differentiation of yersinia pseudotuberculosis bacteria genetic group i - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: Yersinia pseudotuberculosis 413 bacterial strain is recovered from a patient suffering pseudotuberculosis, deposited in the collection of the Russian Science and Research Antiplague Institute Microbe, No. KM 211 and is applicable as a test strain for differentiation of Yersinia pseudotuberculosis bacteria genetic group I. The characteristic of the strain is the content of chromosomal gene of the Y.pseudotuberculosis YPMa/YPMc (urtA/C) superantigen and the YAPI (pilPQ) Yersinia adhesive pathogenicity island gene, the pVM 82 plasmid of molecular weight 82 MDa 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.

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Description

The invention relates to medical microbiology and is an original strain of the causative agent of pseudotuberculosis containing the Y. pseudotuberculosis YPMa / YPMc chromosomal superantigen gene (ypmA / C), the YAPI Yersinia (pilPQ) adhesive “island” gene, plasmids with mol. weighing 82 MDa (pVM 82) and used as a test strain for the differentiation of bacteria Y. pseudotuberculosis of genetic group I.

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 geographic 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-Denved 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 fever, gastrointestinal symptoms and mesenteric lymphadenitis, whereas in 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 t 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 antiphagocytic effect on the immune structures of a macroorganism (Dubrovina V.I., Golubinsky E.P., Borsuk G.I. and author Features of phagocytosis of 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 PCR for the presence of virulence genes such 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-Derived 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 (Messorosh V.G. Biological properties of Yersinia pseudotuberculosis and improvement of laboratory diagnosis of pseudotuberculosis. Abstract of thesis ... candidate of medical sciences, St. Petersburg, 1993, 18 pp.) Used in as a standard for determining virulence, deposited in the collection of pathogenic microorganisms 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 of genetic group I.

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

The obtained strain Y. pseudotuberculosis 413 was deposited in the collection of pathogenic microorganisms of RosNIPCHI "Microbe" KM 211. The strain is distinguished by the content of the chromosomal gene of the superantigen Y. pseudotuberculosis YPMa / YPMc (ypmA / C), the gene for the adhesive pathogenicity of the island of YPP and PAP with a pier. mass 82 MDa (pVM82).

A strain of the family Enterobacteriaceae genus Yersinia, species Y. pseudotuberculosis, isolated from a patient with pseudotuberculosis of a generalized form of moderate severity, St. Petersburg, 2000

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. It contains the chromosomal superantigen gene YPMa / YPMc (ypmA / C), the gene for the adhesive “island” of pathogenicity Yersinia YAPI (pilPQ), plasmid pVM82 (dotO); the chromosomal gene of the “island” of high pathogenicity HPI (fyuA) and the YPMb Yersinia superantigen gene (ypmB) are missing.

Compared with the analogue strain, the presence of a chromosomal pathogenicity factor, YAPI, is also studied in the present invention. The advantage of this study is the use of only the PNR 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. 413 (group I), 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 - PCR results with strains of Yersinia pseudotuberculosis No. 413 lanes (1-5), No. 418 lanes (6-10) and No. 415 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 m.k. / 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 produced by Syntol LLC (Moscow) (table 1)

Table 1 Gene Gene localization Primer Sequence (5'-3 ') PCR Product Length (bp) on the chromosome fyuA HPI Forward CTTTATCCTCTGGCCTTGGG 149 Reverse CTGACAACAGTAGACGAG pilPQ Yapi Forward TATGTTGCTGGAGGCTCAG 569 Reverse GCGAACTATCAGCTATACG ypmA / C Locus YPMa / YPMc Forward CACTTTTCTCTGGAGTAGCG 422 Reverse ACAGGACATTTCGTCA ypmB Locus YPMb Forward TTTCTGTCATTACTGACATTA 268 Reverse CCTCTTTCCATCCATCTCTTA on the plasmid dotO pVM82 Forward ATGATGGATGGTTCACTGGG 2811 Reverse ATTGATTGCTTGAGGTCTTGG

The PCR reaction mixture is prepared using standard Fermentas reagents according to the attached instructions: 2.5 μl of 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 · Na ₂ ) with ethidium bromide staining.

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

It can be seen that strain 413 contains three DNA fragments, 422 bp long, 569 bp and 2811 bp (lanes 1, 4, and 5), which corresponds to the regions of the ypmA / C, pilPQ, and dotO genes. There are no amplicons on lanes 2 and 3.

Example 2. The study of DNA strains of Yersinia pseudotuberculosis.

Using the NDP method using primers for the Yersinia chromosomal genes (fyuA, pilPQ, ypmA / C, ypmB) and the plasmid gene (dotO), DNAs of strains No. 418 and No. 435, which were isolated in a hospital in the treatment of sporadic patients with pseudotuberculosis, were studied. The strain Y. pseudotuberculosis KM 211 was used as a control.

The research results are presented in FIG. 2.

It can be seen that in all the studied strains of Y. pseudotuberculosis, three DNA fragments 422 bp long are present. (lanes 1, 6, 11), 569 bp (lanes 4, 9, 14) and 2811 bp (lanes 5, 10, 15), like the obtained test strain, and, accordingly, they can be assigned to genetic group I. Thus, the fact of the genetic similarity of the studied strains was established, which allows us to judge the circulation of Y. pseudotuberculosis of this genetic groups in the study area.

Thus, the claimed strain is suitable for differentiation of bacteria Yersinia pseudotuberculosis of genetic group I by the content of virulence genes, which will allow to establish the source of infection in the focal point 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 211, for differentiation of bacteria Yersinia pseudotuberculosis of genetic group I.

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