Phage Encapsulation as a Treatment for Vibriosis in Oyster Aquaculture

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

Genome sequences of four Vibrio parahaemolyticus strains isolated from the English Channel and the River Thames

This is the final version. Available from American Society for Microbiology via the DOI in this record.Data availability: Assembled and annotated genomes are publicly available within JGI IMG/M (https://img.jgi.doe.gov/) using the following taxon IDs: V. parahaemolyticus EXE V18/004 (2816332655); V. parahaemolyticus V12/024 (2816332656); V. parahaemolyticus V05/313 (2816332657) and V. parahaemolyticus V05/027 (2816332658). Read data is available on the European Nucleotide Archive under the following accession numbers: V. parahaemolyticus EXE V18/004: ERS3342146; V. parahaemolyticus V12/024: ERS3342147; V. parahaemolyticus V05/313: ERS3342148 and V. parahaemolyticus V05/027 ERS3342149.Vibrio parahaemolyticus, is the lead causative agent for seafood-borne human gastroenteritis. Whilst occurrence has traditionally been uncommon in Europe and the UK, rising sea surface temperatures have resulted in an increased prevalence. Here, we present the complete genome sequences of four novel V. parahaemolyticus strains, isolated from the UK.Natural Environment Research Council (NERC)Biotechnology and Biological Sciences Research Council (BBSRC

A Continuous Culturing Device for use in Bacteriophage Evolution

Over recent years, there has been a steady increase in bacteriophage research. This is in part due to the rise of antimicrobial resistance and in part due to our developing understanding of the role that bacteriophages play in governing microbial communities. Understanding the evolutionary dynamics of both host and phage is an important step towards the applied use of bacteriophages, be it in therapeutic settings, agriculture or aquaculture. Continuous culture techniques are a valuable tool for the systematic exploration of the variety of roles that the physical environment and the conditions within it can play in the long-term dynamics between host and phage. This thesis presents adaptions and extensions to an established turbidostat bioreactor (Gopalakrishnan et al. 2020; 2022) for use specifically in the investigation of both bacterial and viral evolution, using the well-studied model system Escherichia coli and bacteriophage T7 as proof of principle. Briefly, a bacterial host culture is maintained in the exponential phase of growth, using feedback from turbidity measurements. Host cells from this bacterial reservoir are fed on demand into an evolution chamber, which contains a mixed culture of host and phage. Both the metabolic state of the hosts in the reservoir and their influx into the evolution chamber can be controlled, allowing for a variety of evolution experiments using the bioreactor. Further to this, a second project is presented, whereby the bioreactor is adapted for use in a domestic setting, replacing specialised laboratory equipment with commonly found and inexpensive alternatives. This project, carried out in the March 2020 lockdown, describes an example experiment using dried active yeast and has potential application in an education setting. Viral life history parameters are an important method for the characterisation of bacteriophages. Lysis time and burst size, for example, can be used to evaluate the success of bacteriophage reproduction. Therefore, the final chapter of this thesis aims to demonstrate the use of one-step growth curves and Bayesian analysis to estimate the life history parameters of several bacteriophage isolates. Future research may use a combination of the aforementioned experiments to characterise the evolutionary dynamics of bacteriophages and their hosts

Outcomes and characteristics of nonmelanoma skin cancers in patients with myeloproliferative neoplasms on ruxolitinib

Nonmelanoma skin cancers (NMSCs) in ruxolitinib-treated patients with myeloproliferative neoplasms behave aggressively, with adverse features and high recurrence. In our cohort, mortality from metastatic NMSC exceeded that from myelofibrosis. Vigilant skin assessment, counseling on NMSC risks, and prospective ruxolitinib-NMSC studies are crucial.</p