Molecular Biology of Yersinia
Molecular Biology of Yersinia for Diagnostics and Epidemiological Surveillance
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
St. Petersburg Pasteur Institute, Russia, St Petersburg
- Ministry of Health and Long-Term Care / National Yersinia Reference Laboratory, Canada, ON, Etobicoke\nInstitut Pasteur, France, Paris
Project summaryAim of the study. Development of new universal methodologies and algorithms of detection of pathogenic variants of Yersinia spp. and principles of realization of epidemiological surveillance of the diseases they cause based on the molecular biology technologies.
Tasks of the study.
- Creation of collection of clinical strains of Y.pseudotuberculosis and Y.enterocolitica circulating in different regions of Russia that have epidemic significance (Northwest Russia, Siberia/Far East region as well as regions previously not involved in multicenter studies).
- Characterization of the biological properties of the cultures isolated in Russia (serotyping, biotyping, pathogenicity in experiment, enterotoxigenity, Ca-dependent growth, yersiniobactine production etc) and evaluation of their significance to serve as phenotypic markers closely correlating with their pathogenicity. This will allow to evaluate current epidemiological situation in Russia and establish informative phenotypic peculiarities of yersinia strains necessary for determination of their ethiological significance in vitro.
- Selection, synthesis, and assessment of specific oligonucleotides for PCR detection of genetic determinants of pathogenicity of chromosomal and plasmid nature in clinical Yersinia strains (ybt, pgm, hms, irp – Yen HPI) and detection of genetic determinants of other enterobacteria (E.coli and others) that can possibly be transferred between genetically related bacterial species (cnf, hly, ent, pap, eae – PAI-I, -II, -IV, -V, LEE).
- Molecular genetic characterization of clinical strains with epidemic importance circulating in different Russian regions to reveal geographically specific genomic determinants, including “pathogenicity islands”, using DNA-DNA hybridization, polymerase chain reaction, etc.
- Molecular epidemiological typing (RAPD, RFLP, etc) of clinical yersinia strains and analysis of the regional specific features of circulation of human pathogenic variants.
- Comparative evaluation of the revealed genotypic and phenotypic characters of clinical yersinia strains circulating in Russia and analysis of their relationships using statistical and mathematical methods.
- Development of the optimized algorithm of detection of pathogenic Yersinia in different biological substrates based on use both traditional and molecular genetical methods. Increase of informativity and decreasing the time of experiment necessary for modern laboratory diagnostics of the disease and efficient epidemiological control of the circulation the pathogenic yersinia in the environment.
Materials and methods. Biological material from different animal species (wild and experimental) and from human patients, as well as a collection of strains of different origin and from different geographic locations, microbe cell components (DNA, RNA, etc) will be studied.
Methods: microbiological (Yersinia species identification, detection of pathogenicity on in vivo and in vitro models, etc); of molecular genetic (DNA isolation, ribotyping, PCR, sequencing, restriction endonuclease analysis of plasmid DNA); immunological (serologic reactions, isolation and/or synthesis of antigenic components, immunoblotting).
Scientific novelty. As a result of the proposed investigation, the complex evaluation of the current epidemic situation in Russia due to pathogenic yersinia based on genotypic and phenotypic characteristics of epidemically significant Y.pseudotuberculosis and Y.enterocolitica clinical strains from different regions of Russia will be done. The data obtained will provide new understanding of the mechanisms of changing of pathogenicity of opportunistically pathogenic yersinia and enterobacteria as a whole which is necessary for determination of trends of changing of microbial pathogenicity and epidemiological prognostics for yersinia and enterobacteria. The expected results will serve as a basis to decipher genetic mechanisms of pathogenicity as a whole, and those due to “pathogenicity islands”, in particular.
Expected results. This project is a continuation of the long-term St.Petersburg Pasteur Institute activities and is the fundamental research which results can be used as a methodology to study other pathogens.
- Determination of the genotypes and phenotypes of Yersinia strains circulating in the period of outbreak and sporadic occurrence in Russia now will allow to detect and characterize epidemically significant in Russia yersinia variants which detection will provide epidemiological control of the sanitary situation in Russia.
- Complex phenotypic and genetic characterization of the isolated strains will allow to select, deposit and use test cultures yersinia for quality control of the respective culture media, for development of new vaccine and diagnostics preparations, for improvement of the available and development of new antibacterial preparations.
- Determination of the most informative genetic markers of pathogenicity of yersinia and development of optimal methodological approaches of their detection will permit to propose new solutions for development of preparations for express diagnosis of the Yersinia caused diseases (PCR diagnostic kits).
- Genetic characterization of the circulating yersinia strains and analysis of different typing methods based on use of ethiologically significant genetic markers will allow to select optimal methods and provide epidemically significant passportization of strains and database formation of ethiologically significant strains necessary for taxonomic diagnostic and therapeutic tasks.
- A manual for physicians (infectionists, epidemiologists, bacteriologists, etc) will be prepared.
Competence and experience of the participants in the project.
- Specialists in molecular genetics with long-term experience of work with Yersinia, detection of the virulence determinants, who specialized in the laboratories in France and Finland.
- Microbiologists with 5-20-year experience of work with Yersinia who developed the methods and criteria of evaluation of their virulence, diagnostic preparations and methods of indication and identification.
- Immunologists with expertise in the area of antigen-antibody analysis, microbe cell components, including those responsible for virulence and protectivity.
- Epidemiologists with 15-30-year experience of work in the infection foci, in the area of epidemiological surveillance and monitoring of the Yersinia circulation.
- Clinical specialists (adult and children internists) with experience of work with different forms of the disease.
- Other specialists: laboratory workers, project managers, engineers, technical personnel with experience of work in the similar projects. All the specialists have knowledge about construction of the biological weapons and its action on the organism.
The role of foreign collaborators:
- consulting and methodological assistance
- joint studies for DNA sequencing
- information exchange during the realization of the project
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