Properties of Phages for Production of Preparation
Selection of Active Phages against the Y. enterocolitica and Y. enterocolitica Like Strains and Investigation of their Molecular-Biological Properties with an Aim to Construct Medicinal-Prophylactic Polyvalent Preparation
Tech Area / Field
- BIO-IND/Industrial Biotechnology/Biotechnology
3 Approved without Funding
Georgian Academy of Sciences / Institute of Bacteriophage, Microbiology and Virology, Georgia, Tbilisi
- University of Helsinki / Haartman Institute, Finland, Helsinki\nUniversity of Arkansas, USA, AR, Fayetteville\nRobert-Koch-Institute, Germany, Berlin
Project summaryIn the last years a complicated situation has emerged concerning the threat of implementation of biological agents in the bacteriological weapons. The major element of antibacterial defence, which determines effectiveness of it’s use, is clear knowledge of infection proper, its diagnostics, clinic, treatment, and prophylaxis.
Struggle against the yersia-induced infections remains a pressing and complex problem worldwide, because this particular infection is linked to additional social-economic losses.
Y. enterocolitica- and Y. enterocolitica-like strains are widely distributed in the environment and are found in various climatic zones. They have been registered in the rodents, farming- and domestic animals, diary products, vegetables, meat, herbs, fish and sometimes, in drinking water.
The microbes well reproduce at lower temperatures and are maintained for a long time in the environment, as well as in refrigerators and storehouses. Therefore, these objects become the long-lasting artificial reservoirs for the yersiniosis agents. Thus, humans have real opportunity to encounter these microbes, due to wide development of industrial production of the food and their storing, and because of public catering enterprises’ spread.
In 1980-90’s the scientists of the Georgian National Centre of Diseases have determined circulation of various yersinia species in almost all territories of the Republic. The yersiniae as sapronoses and psychrophiles are spread in almost all objects and landscape – geographic zones and climatic conditions. It was established that South of Georgia is a natural focus of yersinia. The Y. enterocolitica and Y. intermedia of biotype I, circulating on the territory of Georgia, are derived mainly from the Yersinia genus. The diseases are manifested mostly by gastro-enterites, colites, food toxic infections and hepatitus.
In the last years mostly the strains with multiple antibiotic-resistance are isolated, which result in formation of heavy and generalized yersinioses of prolonged and relapsing variety, this makes treatment of yersiniosis inefficient even in case of prescription of the antibiotics of choice.
In the meantime there are absolutely no specific means for the yersiniosis prevention. Considering all the above-mentioned, development of the new alternative methods for treatment and prophylaxis of the infection, and creation of highly effective, ecologically clean and safe preparation becomes the urgent problem. The bacteriophages are representing such an effective and safe preparations.
Implementation of the bacteriophages seems to be most promising because they display high specificity of action against the specific infectious agents only.
The great experience in production and implementation of the phage preparations for medication and prophylaxis purposes has been accumulated for decades in the former Soviet Union. Tbilisi Institute of Bacteriophage, Microbiology and Virology, Georgian Academy of Sciences, for many years was a unique leading enterprise concerned with theoretical and applied problems of the bacteriophages. The Institute has developed scientific-methodological basis of creation of the phage preparations for prophylaxis, treatment, and diagnostics of bacterial infections.
Under the supervision of M. Darsavelidze, in a course of the last 25 years, the production technology for medical preventive polyvalent preparations against pyogenic and enteric infections has been developed. The rich collection of pathogenic strains and respective phages (Y. enterocolitica of different serovars, E. coli, Pseudomonas, Staphylococcus, Shigella, Salmonella) has been deposited.
In the 90s, under supervision of M. Darsavelidze, for the first time virulent phages were isolated, which are active against the Y. enterocolitica strains of different serovars. Investigations of the major properties of the clones’ activity have shown that they are characterized by a wide range of action not only against different representatives of the Enterobacteriaceae family, but also against Y. pestis and Y. pseudotuberculosis. We consider that the project realization will provide for creation polyvalent phage preparation, which may be implemented as a mean of antibacterial defence against above infectious agents.
The personnel engaged in the Project has sufficient theoretical background and practical experience in the spheres of microbiology, bacteriophage, immunology, and medicine. They have published a vast number of respective scientific works.
Purposed Project can be ascribed to the category of basic and applied research, as well as to elaboration and demonstration of the technologies.
The aim of the present investigation is selection and investigation of phenotypic and molecular-biological properties of the phages active against the freshly-isolated Y. enterocolitica and Y. enterocolitica-like strains, in order to develop the technologies for production of specific polyvalent medicinal-prophylactic bacteriophages.
The following tasks should be fulfilled in order to solve the above goal:
1. Investigation of biological properties /Morpho-cultural, biochemical and serological/ of different serovars of Y. enterocolitica- and Y. enterocolitica-like strains, isolated from various geographical zones /Georgia, Finland/ and creation of collection. Approbation of the existing collection of the phages against the newly obtained strains.
2. Selection of the new phages active against the received strains of Y. enterocolitica different serovars and Y. enterocolitica like strains, investigation of their activity and stability of lysis.
3. Study of the basic taxonomic trails of separate phage clones. Morphology of the negative colonies, assessment of the phage activity by means of qualitative and quantitative methods, effectiveness of the phage inoculation on different strains, concentration, morphology of the particles with the electron microscopy, mutation frequency, influences of various physical and chemical factors, investigation of the serological properties of the phages, determining the interaction phases with the host-cell, adsorption, latent period, mean harvest.
4. Investigation of molecular properties of the phages. DNA sequencing, PCR Protein studies /2D GELS AND Spectrometry/. Creation of the phages collection.
5. Selection of the tutor-strains and specific phages for the construction of the mother-races. Elaboration of methods for the production of highly effective purified polyvalent bacteriophage.
6. Construction of experimental-laboratory series of medicational preventive preparations of bacteriophages. Elaboration of recommendations for the production of highly active, purified medication bacteriophage preparations.
Prospective application of the preparation:
1. Treatment and prophylaxis in the yersinioses patients; sanation of the recovalescents. Because of existence of the common antigen in the microbes and other representatives of Enterobacteriaceae family, the preparation could be used in treatment of diseases elicited by Salmonella, Proteus, Shigella, Citrobacter, Y. pestis, etc.
2. Treatment and prophylaxis of the farming animals.
3. Processing of hospital quarters, places of public catering, farms and vegetable storehouses.
The collaborators agree to co-operate in scientific-research activities envisaged by the Project, exchange information in the process of its implementation, discuss report materials and in case of necessity provide consultations, discuss the results at conferences, visit the participants of the Project and invite them as well.
The following methods of research will be used in the realization of Project; determination of morpho-cultural traits of bacterial strains; selection of virulent phages and determination of their morphology with the aid of electron microscopy, obtaining and purification of bacteriophages by the method of ultrafiltration, ultracentrifugation in different gradients, etc.; study of the phage propagation cycle; influence of physical-chemical factors on the validity of phages, obtaining of anti-phage serum in rabbits, research of serologic characteristics of phages under the neutralization method; determination of the scope, specific effect of phages on homo- and heterological strains; cross phage-resistance and stability of lysis.
The International Science and Technology Center (ISTC) is an intergovernmental organization connecting scientists from Kazakhstan, Armenia, Tajikistan, Kyrgyzstan, and Georgia with their peers and research organizations in the EU, Japan, Republic of Korea, Norway and the United States.
ISTC facilitates international science projects and assists the global scientific and business community to source and engage with CIS and Georgian institutes that develop or possess an excellence of scientific know-how.