| dc.description.abstract | With our global population expected to increase to as much as 9.8 billion by 2050, strategies for obtaining worldwide food security become increasingly important. The oceans act as a generous resource for reaching our global nutrition targets, yet overfishing in recent decades has caused great harm, including localised population extinction, to fish and shellfish stocks. Aquaculture, the act of maintaining and farming marine or freshwater animal organisms, has become a popular alternative to wild fisheries. However, with a high demand for food sources, comes a move towards more intensive farming practices, whereby denser communities of farmed animals are kept in waters with high nutrient input. Such farming practices can favour pathogenic bacterial communities, which can cause disease in farmed animals and consequently lead to reduced stock numbers. Not only does this affect yield, but there can be further economic impacts to the detriment of those whose livelihoods depend on the aquaculture sector. Traditionally, antibiotics have been used with abundance to treat disease in aquaculture, however overuse and misuse has led to a global rise in antibiotic resistance. This is particularly apparent in aquaculture, where antibiotics can be directly applied to organisms and also, easily accumulate within the water column. Therefore, as the global antibiotic crisis worsens, it has become ever more important to develop novel therapeutic alternatives. One such promising alternative is the use of bacteriophages (phages) – viruses which kill bacteria. However, application of phages requires more research to become commercially viable. Encapsulation of such phages may improve their therapeutic use through increased concentrations during application, improved stability and increased protection. Long term storage of encapsulated phages, e.g. after lyophilisation (freeze drying), would facilitate development of a robust phage library and enable rapid construction of bespoke phage cocktails, whereby distinct phages are combined to combat bacterial resistance. Droplet microfluidics is an emerging field, which can be used for the high-throughput encapsulation of bacteriophages, for use against bacterial infections, not only in aquaculture, but also in clinical settings. Vibrio parahaemolyticus is a highly pathogenic bacterium, capable of infecting shellfish and subsequently cause gastroenteritis in humans. V. parahaemolyticus is commonly isolated from oysters, for example Crassostrea gigas, which is the most commonly farmed species of oyster in the UK, and there is increasing evidence of antibiotic resistance in V. parahaemolyticus. This project aimed to evaluate the use of droplet microfluidics and subsequent freeze-drying in order to encapsulate bacteriophages specific for V. parahaemolyticus, a highly pathogenic bacterium, capable of infecting shellfish and subsequently cause gastroenteritis in humans. V. parahaemolyticus is commonly isolated from oysters, for example Crassostrea gigas, which is the most commonly farmed species of oyster in the UK and furthermore, there is increasing evidence of antibiotic resistance in V. parahaemolyticus. In order to develop of an encapsulated viral library for phage therapy of V. parahaemolyticus, the following four challenges needed to be addressed: (1) isolate novel vibriophages specific for V. parahaemolyticus, (2) develop a novel protocol for the synthesis of monodisperse sodium alginate microcapsules, using microfluidics, (3) encapsulate vibriophages in alginate droplets and (4) use such encapsulated phages to treat V. parahaemolyticus infection of C. gigas. The genomes of four strains of V. parahaemolyticus (EXE V18/004, V12/024, V05/313 and V05/027) were sequenced. In total, 10 dsDNA high quality (category 5) prophage and 5 Inoviruses were detected and manually curated across the four genomes. Furthermore, in this instance the isolation of novel vibriophages was unsuccessful. Despite this, a novel protocol was developed for the synthesis of monodisperse alginate droplets, using a glass microfluidic device. Droplets were synthesised with an alginate concentration of 1 % (w/w) and collected in calcium chloride (CaCl2) solution with a concentration of 2 % (w/w). Bacteriophage T4, vibriophage sm030 and vibriophage sm031 were successfully encapsulated within alginate microcapsules and later lyophilised. Lyophilised droplets containing bacteriophage T4, vibriophage sm030 or vibriophage sm031 were able to cause infection and reduce cell growth in broth cultures of Escherichia coli and V. parahaemolyticus, respectively. More research is needed into phage encapsulation in bacteriophage therapy before its widespread use. | en_GB |
