The development of a phage food additive with the aim to control Salmonella in swine and poultry

Animal experimental design to study the role of phage therapy in acute-non typhoidal Salmonellosis
Co-investigator: Dr. Parameth Thiennimitr, M.D.,Ph.D.

Address: Department of Microbiology, Faculty of Medicine, Chiang Mai University

Species of animal: 6-8 weeks old female mouse (Mus musculus) C57BL6 strain
Protocol (in brief): C57BL/6 mice will be purchased from Nomura Siam International. Mice will be acclimatized for at least 1 week before the experiment day. 24 hour before Salmonella enterica Typhimurium (strain IR715) infection, mouse will be orally gavaged with 20 mg streptomycin sulphate to allow subsequent STM IR715 to establish a colitis. Streptomycin-treated mice will be orally infected with 100 microlitr of 109 cfu/ml STM IR715 solution. Then, 100 microlitr of Salmonella phage solution will be orally gavaged to the infected mice for consecutively 3 days. On day 4 post infection, mice will be euthanized by CO2 and cervical dislocation. Mouse colon content, colon, spleen will be collected for further analysis. Control group (no treatment) of mice will be orally fed with normal saline solution as used as a vehicle for the experimental (phage therapy) groups.
Animal protocol approval committee: All animal experiments conduct at Chiang Mai University will be approved by the Chiang Mai University Animal Care and Use Committee (CMU-ACUC). All animal experiments will be performed in the AAALAC-certified biosafety level (BSL) 2 facility located in Chiang Mai University. Numbers of animal in each group is 7. [Calculated followed Bernard, R. (2000). Fundamentals of biostatistics (5th ed). Duxbery: Thomson learning, 308, level of significance = 0.05)]

Infections caused by antibiotic resistant bacteria currently result in more than 50,000 deaths annually across Europe and the US, with many more people dying worldwide. The emergence of bacterial resistance to all classes of antibiotics is a major global health concern, with the number of attributable deaths worldwide predicted to reach 10 million by 2050. The associated economic, and social loss highlights the pressing need to develop alternative therapeutics. As the identification and development of new antibiotics is slow and difficult, there is an urgent need to explore other viable alternatives. Our work is centred on developing a novel phage based therapeutic approach for Salmonella, which is an important problem worldwide. It is increasingly becoming resistant to antibiotics and both single and multidrug resistant strains have been isolated from the swine and poultry in the farm environment. There is therefore a particular incentive to identify novel therapeutics to prevent and treat infection. Although Salmonella is our focus, the novel timely approaches and tools we will develop will be widely adaptable to target other bacterial gastrointestinal pathogens.

Harnessing the lytic activity and specificity of phages offers a plausible alternative approach to treatment of MDR bacteria, and investment in development of this technology offers an opportunity to have a significant impact in the medium to long term on the quality of life and health. Whilst in the short term, our research will reduce Salmonella from a farm environment and prevent it getting into the human food chain, the longer-term future beneficiaries of this research will be much wider. Clearly the work will have a huge benefit to the export market as currently chicken meat is not allowed to be exported to the EU if it is found to be contaminated with Salmonella. The work will also benefit healthcare providers in Thailand and the UK, and reduce the amount of time nationally that is lost to illness caused by Salmonella infection and subsequent gastrointestinal disorders known as Salmonellosis. Other beneficiaries of the research include the veterinary industry and farmers who are under increasing pressure to not prescribe antibiotics but yet need to treat their livestock.

The proposed research will contribute to advances in knowledge and understanding of how phages can be optimally developed will be used to confirm they have increased efficacy. Again in a context wider than Salmonella, this work will improve efficiency of formulations, and the effective quantitative delivery of phages that target enteric diseases. The beneficiaries of this research include academic and industrial scientists working in the field of biotechnology, and specialists in drug delivery who will benefit from our novel methods and approaches. The lack of methods to allow safe delivery of biological products in the gastrointestinal tract currently limits delivery of all biological therapeutics and our encapsulation technology could also be applied to other orally delivered, biologically based drug and vaccine candidates.

To summarise, our efforts to construct a stable phage preparation will be of benefit to food producers in Thailand and the UK, and in academic and industrial researchers working on developing both prophylactic and therapeutic treatments for a wide range of gastrointestinal infections. Whilst our target country is Thailand, we could in future work extend the phage product development and make it accessible to swine and chicken producers in China, the Philippians and further afield.