The goal of the project. Objectives of this proposal are twofold: (i)identify and characterize the most clinically problematic highly virulent, persistent and drug-resistant strains of Salmonella that cause excessive morbidity and mortality in participating countries, Armenia, Georgia, and Tajikistan; (ii) design phage therapy approaches for efficient control and elimination of these dangerous pathogens from agricultural systems, human food chain, hospitals, and the environment.
The state of the art in the field. Salmonella infections remain one of the leading causes of gastrointestinal disorders in the world resulting in significant morbidity and mortality rates. It estimated that 93.8 million cases of gastroenteritis due to Salmonella species occur globally each year, with 155,000 deaths. In the list of Bioterrorism Agents/Diseases compiled by the CDC, Salmonella is listed in Category B. The principal source for Salmonella infections is the human food chain The key reservoirs for salmonellosis in humans are the farm animals such as poultry, pigs, sheep, and cattle.
The two most common serotypes of Salmonella enterica, S. Enteritidis and S. Typhimurium, are the most frequent causes of acute gastroenteritis worldwide. Clinical presentation of salmonellosis, however, depends on many factors such as the immune status of the host, the serotype of Salmonella and the specificity of the interaction of certain serotypes with the host. In particular, we discovered that the profile of inflammatory responses during the Salmonella infection depend on the serotype of the pathogen. More Salmonella genomic information, however, is needed to uncover the set of genes that drives the differential immune responses that result in different clinical outcome of the disease. Despite the substantial efforts toward the genomics of Salmonella that has allowed a better understanding of the virulence and invasiveness mechanisms, the range of the corresponding mechanisms may be much broader than anticipated, especially in Salmonella populations from geographically distant locations. In this regard, further genomics and transcriptomics analyses of Salmonella isolates collected from distant geographical regions are necessary.
Emergence and rapid dissemination of multidrug-resistance (MDR) among Salmonella limits therapeutic options and poses a serious threat to the public health and represent another problem in controlling of this infection. MDR infections increase morbidity rates and costs to the healthcare systems. We discovered the tendency towards the MDR phenotype and established its association with class 1 integrons in clinical isolates of Salmonella. The contribution of other mobile genetic elements (MGEs) towards the MDR phenotype in Salmonella cannot be excluded and more detailed genetic information is necessary to reveal the MGEs contributing to the appearance of difficult-to-treat infections. Important implication for human health is animal-to-human spread of antibiotic resistance. The second worrying aspect is the evolution of Salmonella towards the acquisition of additional virulence potential. The plasmid pUO-StVR2, for example, provides the Salmonella host with the combination of multiple antibiotic resistance and virulence genes thus potentially causing more serious and difficult to treat infections. Another mechanism of increasing the virulence potential is the acquisition of the corresponding genes from taxonomically distant pathogens.
In the light of the growing problems of antibiotic resistance among Salmonella, and diminishing capabilities to control of infection, other approaches beyond the traditional boundaries of antibiotic therapy are needed to control future Salmonella outbreaks. In this regard, phage therapy has a number of advantages. 1) Unlike a wide range of bacteria targeted by antibiotics, phages are very specific and do not affect other beneficial microbes. This prevents complications such as antibiotic-induced dysbiosis and secondary infections. 2) Phages multiply at the sites where the targets are present thus amplifying the local antibacterial effects. 3) No side effects of phage therapy have been so far detected. 4) Phage-resistant bacteria remain sensitive to other phages and introduction of new phages is a much faster and cheaper process compared to the development of new antibiotics. 5) Phages are efficient in the case of biofilm-forming pathogens that are extremely recalcitrant to antibiotic therapy. 6) Phages may be a valuable source of antimicrobial enzymes such as lysins that are active against difficult-to-treat pathogens. 7) Phages actually limit the evolution and spread of bacterial antibiotic resistance. 8) And finally, the phage arsenal in microbial warfare has recently been expanded by CRISPR-Cas that can be directed towards elimination of most dangerous pathogens.
The impact of the proposed project on the progress in this field. Salmonella infections represent a serious threat for the public health, and we propose to deal with it through the coordinated action of three participating countries. These efforts will be directed at analysis and identification of the most problematic Salmonella strains and elaboration of novel phage therapy strategies to eliminate the reservoirs of these strains to prevent the future Salmonella outbreaks.
Expected results. The project consolidates efforts of experts from Armenia, Georgia, Tajikistan, and six collaborating countries (Denmark, Belgium (two institutions), Ireland, The Netherlands, Sweden, and United Kingdom), to detect the most clinically problematic Salmonella strains in participating countries based on clinical presentation of salmonellosis and drug resistance profiles and provide novel phage therapy for efficient and targeted elimination of the most virulent, persistent and multidrug-resistant (MDR) Salmonella strains. This proposal will use sequence analysis of Salmonella genomes to uncover the role of genes differently represented/expressed in the genomes of various clinically problematic Salmonella serotypes during infection and explain the differential induction of host’s inflammatory responses. This work will answer the question how varies the pathogenic potentials of genetically close Salmonella serotypes. Sequencing and annotation of clinically problematic Salmonella strains will provide information on the genetic background of MDR phenotypes and genetic kinship of the human Salmonella enterica isolates circulating in participating countries. More importantly, the phage therapy approach targeting these dangerous pathogens will be elaborated. This powerful tool will be directed towards the elimination of most virulent MDR Salmonella pathogens from the natural reservoirs such as agricultural animals to prevent their entry into the human food chain and cause fatal disease. The resulting phage formulae efficiently killing Salmonella pathogens will be patented and commercialized.
Meeting ISTC Goals and Objectives. The project is of great value and importance from that point of view that presents to the scientists of the Institute of Molecular Biology NAS RA, which were earlier involved in the defensive activity of the former CIS, a possibility for the health-oriented research. The project cooperates to the integration of the scientists from Georgia, Tbilisi, namely with Dr. Nina Chanishvili from the Eliava Institute of Bacteriophage, Microbiology and Virology, and Prof. Farida Tishkova, Tajik Research Institute of Preventive Medicine, Dushanbe,Tajikistan. The project is directed to the investigation and designation of recommendations for solving health-related problems in the field of infectious diseases, particularly salmonellosis, and the development of innovative phage therapy strategies against the most virulent and antibiotic-resistant Salmonella strains.
Scope of activities. The project is planned for two years. The basic research will be carried out on Salmonella strains isolated from patients with complicated and hard-to-treat salmonellosis in participating countries to design the novel phage therapy for control and efficient and targeted elimination of the most virulent, persistent and multidrug-resistant (MDR) Salmonella strains. The specific tasks of this proposal are: 1.Consolidation of efforts of experts in antimicrobial resistance and phage therapy in Armenia, Georgia, and Tajikistan as well as in collaborating countries; 2. Identification of the most clinically problematic strains of Salmonella in participating countries based on clinical presentation of salmonellosis and drug resistance profiles; 3. Sequencing and annotation of strains identified in objective 2; 4.Screening the Salmonella bacteriophage collection for the representatives that are most efficient at killing these Salmonella strains; 5. Sequencing and annotation of bacteriophages identified in objective 4; 6. Designing and testing in vitro therapeutic phage formulae that are most efficient at eliminating the most virulent and antibiotic-resistant Salmonella strains; 7.Commercialization of the phage formulae elaborated during the project.
The role of foreign collaborators. The role of foreign collaborators will be in assessment of the project quality, coordination of efforts, help with technical challenges, monitoring the progress of the project. Routine communication will be primarily through the electronic mail and Skype videoconferencing. The cooperation will be coordinated also through the reciprocal visits, which will allow discussions, and preparation of results for publications. The collaborators in this project are Dr. R. Aminov from the Technical University of Denmark and Professor Peter Mullany from University College London (United Kingdom), who are the leading world experts in antibiotic resistance. The world leading phage biology experts are represented by: Professor Anders Nilsson from Stockholm University (Sweden), Professor Mario Vaneechoutte from the Ghent University (Belgium), Dr. Aidan Coffey from Cork Institute of Technology (Ireland), Dr. Jean-Paul Pirnay from Queen Astrid Military Hospital (Belgium), Professor John van der Oost from Wageningen University (The Netherlands), and many other informal supporters from Europe, North America, and Japan. They will provide the necessary level of expertise and knowledge in the area to achieve the tasks in this project.
Technical approach and methodology. The methodology proposed for this work includes: (i) identification and collection of clinically virulent Salmonella strains from diverse regions, (ii) determination of their antimicrobial resistance profiles, (iii) sequence analysis and annotation and comparative genomics of Salmonella, (iv) identification of the most efficient and reliable phages targeting these strains, (v) sequence analysis and annotation and comparative genomics of these phages, (vi) design and in vitro tests of phage formulae, (vii) patent protection and commercialization of the formulae obtained during the project.
