The aim of the Project: Obtaining of peptides with immune regulatory activity for creation of new immunomodulators.
The prevalence of especially dangerous infections is one of the major problems in developing countries. In recent years antibiotic resistance of gut microbiota pathogens has been considered a problem due to the extensive use of classical antibiotics in treatment of human and animal diseases. Resistance to antibiotics is an increasing, global problem for public health. In the WHO European Region, the resistance of some pathogens now reaches over 50 % in some countries and new resistant mechanism are emerging and spreading rapidly. At the same time, the problem has escalated over the past two decades as the prevalence of antibiotic-resistant bacteria. Multi-drug-resistant strains have emerged in many species that cause disease in humans and animals. The main problems that may emerge in the cattle and poultry industry are antibiotic resistant microorganisms on the one hand and the control of treatment of intestinal infections without antibiotic use. In order to improve the health of animals and birds need to regularly monitor immune status of organism usage of antimicrobial agents with caution and, at the same time to exclude ineffective application of antibiotics.
Currently, particularly important to determine the value of the immune system in its fight against infectious diseases. It is known, that disease intrahospital infection often confirmed with individuals having weak immune systems, so it is especially important to develop the ways to increase the immunological status of the organism. One of the promising approaches would be to creation of the bank (collection) of peptides with immune stimulatory properties.
Research that has been ongoing for more than 60 years to identify novel antibiotics, has led to one of the most promising new concepts in antimicrobial technology – the use of peptides as antimicrobial agents. One of them, antimicrobial preparations (AMP), termed bacteriocins (BCN) are natural peptides that have bactericidal effect. AMPs represent bactericides (cell killers) instead of bacteriostatics (bacteria growth inhibitors) and, therefore, might not induce the resistance. AMPs have a broad range of activity and are excellent candidates for development of new prophylactic and therapeutic substances to complement or replace conventional antibiotics.
At the Laboratory of Antimicrobial preparations of SPC “Armbiotechnology” NAS RA, more than 250 endemic cultures of lactic acid bacteria (LAB) from different traditional for Armenia dairy products from rural households were isolated. The morphological and physiological features, probiotic properties, specific growth rate and antimicrobial activity of LAB were studied. Strains of bacteria were identified by the methods of genotyping including API test, 16S rRNA sequencing and RAPD PCR: they were presented by Enterococcus faecalis BTK 207, Enterococcus faecium BTK 64, Lactobacillus plantarum BTK 65, Lactobacillus rhamnosus BTK 109, Lactobacillus plantarum BTK 66, Pediococcus pentosus BTK 28, Streptococcus lactis 103. Microbial cultures have been deposited at Department of Center of Microbial Depository (CMD) under SPC “Armbiotechnology” NAS RA.
The performed laboratory studies demonstrates that purified supernatants obtained from the culture liquids (Cl) after the cultivation of several LAB possessed a different spectrum of growth suppression of various pathogenic microorganisms. The peptides obtained from Cl (purification by HPLC method) Lactobacillus rhamnosus BTK 109 (BCN11470 Dа and BCN 2670 Dа) and L.acidophilus 1991 (BCN 1100 Dа) shown different antimicrobial action against gram-negative and gram-positive pathogens, such as Salmonella sp., E.coli, Proteus mirabilis Pasteurella spp., Clostridium spp, Streptococcus spp., Staphylococcus aureus, Shigella spp., Yersinia enterocolitica, Bacillus cereus, Listeria monocitogenes spp., as well as especially dangerous pathogens Y.pestis V.cholerae Y.enterolitica, Francisella tularensis. Analysis of scientific articles shown, that there is no data on natural AMPs’ effectiveness against EDP that cause diseases such as brucellosis, yersiniosis, cholera, tularemia, anthrax, and plague.
Partially purified preparation of bacteriocin from L.acidophilus 1991 successfully implemented for the prophylaxis of animal infections (Salmonella sp.) in the bird and swine farms.It was shown overweight of animals, absence of mortality. At the same time during re-infection by Salmonella sp., the absence of disease was observed, which may indicate an increase of the immune status of the animals. So, the continued development of new classes of natural antimicrobial agents, possessing antibacterial activity, has become of increasing importance for medicine and veterinary.
Antibiotic resistant pathogenic strains were isolated by us from infected patients in Infections Hospital and from different food products in the National Bureau of Expertise (Yerevan, RA). It was shown, that out of the 23 strains 21 (91.3%), multi-resistant to the action of from 2 to 9 antibiotics, studied strains showed high sensitivity to fluoroquinolones and high resistance to an beta -lactams.
The differences of sensitivity of antibiotic resistant hospital strains to bacteriocins from L.rhamnosus BTK 109 and L.acidophilus1991 strains was shown. It is suggested, that this may be due to the nature of antimicrobial substances (peptides) synthesized by LAB strains belonging to different genera and species and due to its structure features and amino acid composition. For this purpose, the investigation of the structure of bacteriocins isolated from seven strains of studied LAB, comparison of the spectra of growth inhibition of antibiotic resistant bacteria, establishing of relationships between structure and antimicrobial and immune regulating properties of peptides is of interest. The sequence of amino acids of most perspective peptides with low molecular weight will be carried out and the works on getting their synthetic analogues will be done.
During the creation of probiotics a major problem is selection of strains which are specific to human intestinal microbiocenosis. However, now the concept of probiotics expanded and include bacteria isolated from dairy products. That were shown by our studies. Therefore, the obtained data also allow to create the associations of perspective strains LAB with predetermined probiotic, antimicrobial, immune modulatory (stimulating) properties to obtain new functional food products.
Scientific and Production Center “Armbiotechnology” NAS of Armenia is actively engaged into the study of bacteriocins from LAB within the realized ISTC-GIPP Projects A-1957 “Isolation and characterization of probiotics selectively growing on milk oligosaccharides” (2012-14), ISTC-GIPP Projects A-1866 “Natural biological substances (peptides) with bactericidal properties toward various pathogens” (2010-13).
Researchers of Center for Life Sciences Nazarbayev University participating in the project have extensive experience in the study of probiotic cultures and development probiotic food supplements, including the isolation of microorganisms from different ecological niches, the study of colonization potential, functional activity, including antioxidant, radioprotective, DNA - protective properties. Also, researchers are experienced in conducting preclinical and clinical trials of probiotics, over the past 5 years they have published 6 articles in international journals, 3 books (2011, 2012), and 10 patents.
