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The World Health Organisation (WHO) reported that 600 million foodborne cases of illness occurred annually, with 420,000 deaths. The emergence of antimicrobial-resistant (AMR) foodborne pathogens is one of the world's major health concerns that need addressing. The three foodborne pathogens targeted in this project are Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium and Shigella flexneri. They are important gut pathogens, causing diarrhoea disease worldwide, and AMR strains of these pathogens are putting millions of lives at risk. The use of bacteriophages (phages) is being considered as an alternative to antibiotics to combat AMR. Bacteriophages are highly specific in respect to their host, which makes them an attractive antibiotic alternative, which will result in minimal disruption to other commensal bacteria. A transducing unit system based on bacteriophages P1 and P2 for the delivery of an antimicrobial Cas9 nuclease system was developed in this study. Cas9 can be programmed to target specific DNA sequences in a bacterial genome, inducing double-strand breaks, which lead to cell death if not repaired. The use of the Cas9 nuclease as an antimicrobial is an attractive approach for the precise elimination of pathogens. To expand the potential biocontrol and therapeutic applications of these phage-based transduction systems, I have modified the host range of both phages P1 and P2. The host range of P1 has been expanded and retargeted to a novel host S. Typhimurium through the use of an O-antigen degradation enzyme (phage P22 tailspike, Gp9) as an adjuvant. The exogenous application of the P22 tailspikes was able to sufficiently digest the O-antigen on the cell surface of S. Typhimurium to allow P1 transduction. Although using the purified P22 tailspike as an adjuvant permits P1 transduction in S. Typhimurium, this approach relies on both the P22 tailspikes and P1 to co-localise for the transduction to occur. In an attempt to improve the transduction efficiency of P1 into S. Typhimurium, a polyvalent phage P1 was created. Here, the phage P1 was engineered to have 2 different tail fibers, wild type (WT) P1 tail fibers and chimeric P1-P22 tail fibers. The P1-P22 chimeric tail fibers were expected to have an O-antigen degradation activity, which would degrade the lipopolysaccharide (LPS) and allow P1 WT tail fibers to bind to the core LPS and transduce into the cell. Various designs of P1-P22 tail fibers were constructed, but no LPS degradation activity was detected. The idea of a polyvalent P1 phage was not explored further. Another bacteriophage that was investigated in this project is phage P2. The P2-based transducing unit production protocol was optimised to produce high titers. In addition, the P2 transducing units were engineered for the delivery of a Cas9 antimicrobial, which was shown to be efficiently packaged in the capsid to generate a high yield of transducing units. The host range of phage P2 has also been modified via tail fiber engineering to recognise a novel host, E.coli O157:H7, and improve its transduction efficiency in S. flexneri M90T. The chimeric tails allow higher Cas9-mediated killing levels in E. coli O157:H7 and S. flexneri M90T compared to WT P2 tail fibers. The work presented in this project demonstrates the proof-of-concept of using P1 and P2-based transducing units as pathogen control agents. The P2-based transducing units containing a chromosomal-targeting cosmid were able to efficiently kill important gut pathogens, S. flexneri 2457T, S. flexneri M90T and E. coli O157:H7. Engineered bacteriophages can serve as an important tool for antimicrobial delivery and have many potentials in clinical and biocontrol applications.

A bacteriophage P1 phagemid system allowed efficient packaging of a DNA-sequence specific, Cas9 antimicrobial into transducing particles, which could be used to infect and kill pathogenic bacteria, such as Shigella flexneri. Although the transduction of a cas9 phagemid yielded significant antimicrobial effect on S. flexneri, the phagemid lysates produced a significant non-Cas9-mediated cytotoxic effect, which might be attributed to the presence of wildtype P1 phage and/or other cytotoxic factors in phagemid lysates. Treatment of phagemid lysates with a low concentration of polythene glycol (PEG) reduced the titre of wildtype P1 phage and led to a lower cytotoxicity of phagemid lysates on S. flexneri cells. Mutations introduced into the P1 lytic origin of replication (oriL) inhibited its lytic stage DNA replication, yet the mutant was incapable of producing phagemid transducing particles. Deletion of the DNA packaging site, pac, from the P1 genome significantly reduced the titre of wildtype P1 phage. A Δpac mutation, however, did not reduce the cytotoxicity of phagemid lysates significantly, when used for treatment of S. flexneri in vitro and in a zebrafish larvae infection model. An alternative P4 cosmid system allowed efficient packaging of cas9 construct into transducing units without the contamination of host and/or phage DNA. Contrastingly, treatment of S. flexneri with P4 cosmid lysates gave negligible cytotoxic killing of the bacterial cells, suggesting that the non-Cas9-mediated killing effect might be attributed to impurities associated with the P1 phagemid transducing units and/or due to inherent properties of P1 transduction. Nevertheless, the versatility of the P1 phagemid system allowed an inducible Cpf1-mediated, vector curing function to be incorporated into the phagemid while retaining its Cas9-mediated antimicrobial effect on S. flexneri cells. Furthermore, the P1 cas9 phagemid was compatible with the ΔnadC autotrophy complementation system, providing antibiotic-free maintenance and selection of the modified vector in ΔnadC E. coli. Taken together, the phagemid and/or cosmid system that produces pure transducing particles might be the key to achieving a high Cas9 antimicrobial effect on S. flexneri cells with negligible lysate cytotoxicity.

Antimicrobial resistance (AMR) is set to be the next global pandemic with AMR related deaths set to outgrow cancer deaths by the year 2050. Increasing resistance to the traditional arsenal of antibiotics is expanding at an alarming rate and novel antimicrobial therapeutics are urgently required to tackle this emerging issue. Proteus mirabilis and Pseudomonas aeruginosa are two bacterial species commonly associated with urinary tract infections in humans. To contribute to the progression of novel antimicrobials against P. mirabilis and P. aeruginosa, this thesis was based on the isolation and utilisation of bacteriophages and bacteriophage derived products to help treat biofilms caused by both bacteria. The isolation of phage PM-CJR showed promising antimicrobial potential against P. mirabilis due to the production of depolymerase enzymes. The thesis describes the identification and utilisation of both the whole phage and its tail-spike protein with the pectate lyase activity to treat P. mirabilis biofilms. The phage and the tail-spike protein were assessed in a range of different in vivo and in vitro assays to demonstrate their antimicrobial and antivirulence properties against P. mirabilis biofilms. This thesis also studies the combinational treatment of phage and cold atmospheric plasma for the treatment of P. aeruginosa biofilms and establishes the most effective order or application of each antimicrobial for the most effective treatment option against P. aeruginosa biofilms. We successfully showed that the use of CAP followed by exposure to P. aeruginosa phages is the most effective order of application for eradication of P. aeruginosa biofilms.

The emergence of multi-drug resistant bacteria has created an urgent need for new and alternative viable antibacterial therapies. With the antibiotic development pipeline for new classes of drugs experiencing a decline in new successful treatments coming to market, a focus towards evaluating alternatives has increased significantly over recent years. Specifically, bacteriophage (phage) therapy has sparked renewed interest with the application of living, bacteria specific viruses for infection control. Other narrow spectrum antibiotics comprise bacteriocins and endolysins, both of which have proved promising in vitro. There are major advantages of phage and protein antibiotic therapy in comparison to using conventional broad-spectrum antibiotics, such as the ability of phages and bacteria/phage derived proteins to evolve alongside the target bacterial strain and the possibility of microbiome engineering. However, limitations include the sensitivity of phages and proteins to processing conditions encountered during their manufacture as well as the detrimental impact on viability upon exposure to environmental stresses experienced e.g. during transit to the site of infection. These include acid and enzyme exposure in the stomach for gastrointestinal applications and the impact of the host's immune system on phage and protein pharmacodynamics. Hence, there is an imminent need to explore encapsulation approaches of each potential antibiotic into targeted delivery and controlled release formulations. In this PhD thesis, initial research into bacteriophage processing enabled high titres (1010 PFU/ml) of phages to be produced using a batch mode of production (shake flasks). The process of membrane emulsification was selected for encapsulation of enteric phages building on previous published work using this process. Firstly, the parameters of droplet production were investigated to confirm the suitable parameters required for the controlled production of droplets with a mean diameter of ~100μm. Subsequently, an E. coli specific Podovirus was encapsulated using three different pH-responsive formulations due to incorporation of either Eudragit L100-55, L100 or S100 polymers. Phage encapsulation in each formulation was tested to confirm different pH-dependent release characteristics suitable for targeting different sections of the GI tract (e.g. human duodenum, jejunum, ileum, and colon). The encapsulated phages in the microcapsules withstood acid exposure and showed good stability upon storage over 6 months under refrigerated conditions. Membrane emulsification in pH-responsive formulations was used to encapsulate an E. coli specific bacteriocin (colicin Ia and E9). Initially, a process for the overexpression and purification of colicin was developed yielding protein at a suitably high concentration (100mg/ml) for encapsulation. Incubation time prior to addition of IPTG, and IPTG concentration were parameters that were varied enabling sufficient amounts of colicins E9 and Ia (~80-100mg) per 5 litre batch to be produced. Subsequently, the colicins were encapsulated using membrane emulsification and tested in vitro to confirm protein yields within the microcapsules. An in vivo murine E. coli colonisation model was utilised to evaluate the delivery of encapsulated colicin to the mouse caecum as a proof-of-principal study. Significant reductions in CFU were observed in the colicin treatment group of mice (in comparison with the untreated group) verifying that the encapsulated colicin in the pH-responsive microcapsules reached the site of infection. A significant proportion of mice within the treatment group displayed no E. coli colonisation following 3 days of treatment. Looking from a different perspective, antibiotic resistance genes are passed through the food chain and are specifically exacerbated within the meat production industry. Antibiotics are routinely overused worldwide during meat production to prevent the spread of disease through animal populations and, in some countries, continue to be used as a growth promoter. The use of phages added to animal feed could help facilitate a slowing down of the rate of resistance gene emergence, reducing carriage and transmission of AMR bacteria, as well as for the successful treatment of enteric bacterial infections. Incorporating phages in animal feed requires consideration of processing stresses such as hot extrusion encountered during animal feed pellet production. Phages were formulated into core-shell pH-responsive capsules with the aim to design capsules that allow the phages to withstand wet thermal heat treatment and extrusion. The encapsulated phages withstood exposure to 95⁰C for up to 120s, in comparison with just 15s without encapsulation. The encapsulated phages in the core-shell capsules survived acid exposure for 2 hours at pH 1 with no significant change in phage titre. Finally, a novel phage endolysin with specific lytic activity against clinical C. difficile strains was encapsulated in pH-responsive capsules to protect against gastric acidity and enzymatic damage. The endolysin displayed lytic activity against three strains of C. difficile in vitro using turbidity and CFU reduction assays. Concentrations as low as 3.8μg/ml enabled a decrease in optical density and a modest reduction in viable bacterial counts. A concentration dependent rate of lysis was observed, with higher concentrations facilitating rapid rates of bacterial killing. The in vivo activity of the endolysin was evaluated using a Galleria Mellonella infection model in which the larvae were force-fed C. difficile followed by endolysin treatment after hours of infection. The G. mellonella Health Index Score was utilised to determine the severity of C. difficile infection at discrete timepoints throughout the infection period (Loh et al., 2013). Results indicated higher larvae survival rates after 2 doses of endolysin treatment as well as improved Health Index Scores, suggesting a less severe infection. The results presented in this thesis demonstrate a number of different encapsulation approaches and pH-responsive formulations suitable for a range of novel biotherapeutics currently under development as alternatives to conventional antibiotics.

Food waste is an issue of global importance. The ever-growing population on Earth combined with a finite amount of resources means that the protection and production efficiency of food is paramount to the ability to feed people without waste. Food waste is primarily an issue for economically developed countries where consumers have the luxury of choice when it comes to selecting the food products they purchase. There are several factors that influence the consumer's perception of a product: how the food itself looks, the smell, and the taste. Any deviation from what is considered 'normal' or 'safe' by the consumer can lead to the disposal of food that is safe for human consumption, and thus wastage of nutritional resources. Food spoilage microorganisms are predominantly responsible for changes in look, smell, and taste of a product, but not all spoilage renders food unsafe. Several spoilage species are associated with the production of off-odours in a variety of fresh meat products, and their presence on meat, though harmless, causes a significant of food waste. Due to restrictions in food processing, there are limits on what can be done to meat to remove bacteria. As such, the practical application of bacteriophages in meat production is a promising solution to reducing food waste. This is due to their specificity, organic source, growing government approvals of bacteriophage-based products, and proven efficacy in the removal of pathogens from food. This project assesses the suitability of bacteriophages for application in fresh vacuum-packed pork products to reduce growth of target spoilage bacteria and production of off-odours. The odour of vacuum-packed pork sampled across a seven-month period is characterised and the bacteria responsible for the off odours are identified. In addition, the effect of oxygen environment on phage infection efficacy is investigated, providing novel insight into bacteriophage/host interactions.

Bacterial antimicrobial resistance represents a significant challenge to global medicine. Bacteriophage therapy is receiving renewed attention as a promising alternative, or supplement, to conventional antimicrobials, however, bacteriophages do not penetrate eukaryotic cells well. This limits their use against intracellular bacterial pathogens, such as Mycobacterium tuberculosis and Rhodococcus equi (R. equi). Both these pathogens reside and grow within alveolar macrophages in lungs, infection requires long treatment and antimicrobial resistance is of concern. One approach to overcome this limitation of bacteriophages, is delivery within liposomes, a well-established method for targeting drugs to sites of infection, including within eukaryotic cells. The aim of this project was to formulate and test liposome-encapsulated phage preparations for activity against intracellular Actinomycete pathogens. Eight novel R. equi bacteriophages, including 4 which infect R. equi at 37°C, were isolated from soil samples collected at equine studs with a previous history of foal R. equi infections. Genomic and biological analysis of these bacteriophages revealed that these Siphoviruses share low genomic similarity. Based on size, host range, and kinetic features, R. equi phage AAM1 was selected for liposome encapsulation experiments. Different liposome lipid formulations and phage encapsulation efficiency were assessed. Liposome encapsulated bacteriophage AAM1 had higher efficiency than free bacteriophages in treating R. equi infecting J774A.1 macrophages. Prophylactic treatment of macrophages with either bacteriophage preparation was unable to prevent the establishment of R. equi infection. Although free mycobacteriophage TM4 had no effect on M. bovis BCG when applied after uptake into THP-1 macrophages, application of liposome encapsulated TM4 resulted in a significantly higher numbers of intracellular BCG. In conclusion, R. equi bacteriophages with therapeutic potential have been isolated. The preliminary evidence of efficacy of preparations of liposome encapsulated R. equi bacteriophage AAM1 in treating intracellular R. equi, supports the need for further studies and optimisation of liposome formulations.

Bacteriophages (phages) are viruses that infect bacteria. Although it is clear that phages may help the host immune system to kill bacterial pathogens, many details of bacteria-phage-host immune interactions remain poorly understood. Bacterial outer membrane vesicles (OMVs) are one of the virulence factors of Gram-negative organisms. OMVs are spherical membrane-enclosed microparticles produced during bacterial growth. During bacterial infections, OMVs are recognized by the host immune system and participate in the elicitation of immune responses. OMVs have been found to interact with bacteriophages in a host-specific manner. The effects of such interactions on the biological functions of OMVs remain largely unknown. In this project, research into the immunological consequences of bacteriophages interacting with OMVs was assessed by investigating whether OMV-phage interaction affects the inflammatory response elicited by OMVs alone. An important methodological development from this project was to optimise a protocol to isolate and purify OMVs from E. coli. OMVs were isolated in a highly pure form. They were interacted with purified E. coli specific phages. OMVs, phages and OMV-phage mixtures were then interacted with macrophage cells, and it was found that the OMV-phage interaction led to a reduction of the pro-inflammatory response elicited by OMVs alone. This suggests that phages may impact bacterial-host immune system interactions not only by killing the pathogens, but also by altering host responses elicited by the conserved bacterial virulence factor, OMVs. Furthermore, preliminary results revealed that interactions of a bacterial lysogen with macrophages or whole blood samples resulted in the induction of prophages from lysogenized bacteria. This may suggest that during the interaction of bacteria with the host immune system, some bacteria may be killed not by the immune assault per se, but by the induced prophages. Three bacteriophages infecting K. pneumoniae were isolated from sewage samples by the traditional method of phage isolation. Genomic and biological characterisations were performed on two of these phages. These phages were used in this current research and may be used in the future to investigate their impact on the immunological responses elicited by Klebsiella OMVs, once a methodology to isolate sufficient quantities of OMVs from this pathogen is developed.

In nature, bacteria are frequently exposed to predation by bacteriophages and protozoa. These two microbial groups were described as the main causes of bacterial mortality. Yet, bacteria were found to co-exist and thrive in the presence of both predators. This study was established to investigate whether the presence of Acanthamoeba can protect bacteria from killing by bacteriophages. Gentamicin protection assay was used to determine the ability of P. aeruginosa WB1 and E. coli 4s to intracellularly survive within Acanthamoeba castellanii cells. Later, bacteria retrieved form gentamicin protection assay were subjected to phage infection. Phages were applied in a form of single and combination treatments. Results have indicated that P. aeruginosa WB1 and E. coli 4s were not able to intracellularly survive within the amoebal cells and were digested by 99%. And, single-phages were effective in a single-enemy system (phage-bacteria system) but were not effective in multiple-enemies system (in Acanthamoeba presence), while, phage combinations showed higher efficiency in eradicating bacterial numbers in both systems. In addition, this study has investigated the ability of phages to internalise and survive within Acanthamoeba cells. Two experiments were performed, first, the ability of phages to survive inside Acanthamoeba cysts was assessed. Non-nutrient agars were seeded with bacteria infected with phages, then, Acanthamoeba trophozoites were introduced and incubated at room temperature for two weeks to induce encystation. Later, cysts were exposed to acid (2% (v/v) HCL) treatment, UV radiation and extreme heat to assess Acanthamoeba ability to protect phages from hostile conditions. Second, phages (without bacterial hosts) were co-cultured with trophozoites and exposed to UV radiation and extreme heat. Results have indicated that phages were able to intracellularly survive within Acanthamoeba cells. Phages were found more resistant to adverse conditions once trapped within Acanthamoeba cysts, as well as, in the presences of amoebal trophozoites.

Bacteriophages are viruses able to infect and kill their bacterial host upon replication within it. They are thought to be the most abundant organism on earth, since their population exceed the one of bacteria (about 1030) by a factor of 10. In many cases, the host range where a given bacteriophage is active is so narrow that it is usually specific to one species of bacteria. In the early 1920s, phage were used to treat bacterial infections in eastern Europe and spread to western companies that began to commercialize typhoid and urinary-tract infection therapies. Soon after, because of penicillin discovery, researchers abandoned this therapy for antibiotic treatment, mostly due to phage susceptibility to the immune system and other unknown behaviours. Due to the overuse and misuse of antibiotic drugs, antibiotic resistance fast become a serious problem of global concern, currently with no real solution, which represents a threat to humanity leading to a post-antibiotic era if no counter-measures and alternative strategies are undertaken. In the last decade, bacteriophage application for the treatment of bacterial infections has re-gained huge attention. However, phages delivered in buffer solutions are suboptimal, since they are not shielded from host defences that inexorably hinder phage activity. Therefore, for effective in vivo applications, e.g. crossing the acidic environment of the stomach or infecting intracellular pathogens, phages need to be delivered to the site of infection, for example via encapsulation. Encapsulation in targeted delivery systems will help phages to reach the target with high specificity, minimizing losses, undesired adverse side effects, and limiting the likelihood of emerging "super bacteria". In the last decades, a considerable variety of stimuli-responsive micro/nanocarriers have been designed. Such systems show a transition to the supramolecular structure or in their chemical structure in response to given stimuli. A wise choice of the stimulus that triggers the variation allows releasing of their cargo in a spatial- and temporal- controlled fashion. The stimuli can be either external or internal and most commonly are thermal variation, pH, enzyme concentration, redox potential, magnetic field, ultrasound intensity and photo sensitivity. In this thesis, we study the encapsulation of bacteriophages in lipid carriers able to deliver and release the cargo in a controlled manner. Liposomes are biodegradable non-toxic micro/nano vesicles composed of a lipid bilayer, which encloses an aqueous core that offer many advantages in medicinal applications. It was demonstrated that phage encapsulation in liposomes improves stability. Indeed, when inside the aqueous core of a vesicle, the thermodynamics of the system is advantageous for phage storage. In this doctoral thesis we have assessed the encapsulation of two model phages, an Escherichia coli T3 podovirus (size ~65 nm) and a myovirus Staphylococcus aureus phage K (capsid head ~80 nm and phage tail length ~200 nm). Encapsulation in liposomes was carried out by producing the vesicles by alcohol injection method in a capillary microfluidic device, in the presence of the cargo. We generated liposomes having mean sizes between 100-300 nm and the encapsulation yield of T3 phages was 109 PFU ml-1 and for phage K was much lower at 105 PFU ml-1. The encapsulation efficiency was affected by the aggregation state of the virions. Furthermore, we discovered that phage K is able to interact with the lipid bilayer resulting in phages bound to the outer membrane of the liposomes instead of being encapsulated inside them. We utilised the encapsulated phages to determine whether 1) they are internalized by human macrophages in higher number compared to free phages and whether 2) they are able to reach the cytosol were intracellular pathogens reside. We discovered that, regardless the formulation of liposomes, encapsulated phages are intracellularized more efficiently in comparison to free phages. However, following uptake, liposomes and encapsulated phages are trafficked to the lysosome, where they are irreversibly digested. Therefore, we envisioned a different approach: a smaller antimicrobial agent than phages, namely a phage lysin, was co-encapsulated with a hemolysin able to make pores in the endosomal membrane. We observed that the co-encapsulated of the two proteins effectively escaped lysosomal degradation and defeated intracellular S. aureus. We expect this approach to be easily extended to phages and we suggest further perspectives and concrete future experiments. In this doctoral thesis, we made significant advances in the encapsulation and intracellular delivery of phages for antimicrobial treatment, and we obtain promising results that pave the way to the actual utilization of phages for medical treatment of infections, especially of antibiotic resistant ones.

Antimicrobial resistance poses a major problem to health and new alternatives to antibiotics are required. Bacteriophages are viruses able to selectively kill bacteria and they can be exploited to treat infections. Another alternative to antibiotics that can be used to treat bacterial infections are bacteriocins, which are proteins produced by bacteria, meant to kill other strains competing for the same nourishment source. Future industrial demand for large quantities of bacteriophages and bacteriocins imposes the development of a scalable production platform. In this PhD thesis, research on bioprocessing of bacteriophages K and T3 and one bacteriocin belonging to the subcategory of colicins, E9, has been conducted. The aims were to find the parameters involved in production of phages and colicin E9 using shake flasks and then exploiting them to perform continuous production of both phages and colicin E9 and showing the different outcomes when using complex medium (LB) and a synthetic medium (SM) using glucose as only carbon source. For the colicin E9 bioprocessing, tests for expression were carried out to measure when and for how long to induce for optimal protein production and the best conditions for production were an induction of 3 h with 1 mM of IPTG in LB medium and >10 h of induction with 1 mM of IPTG in SM. These parameters were used for continuous production of colicin E9 that was carried out using a chemostat. Optimal dilution rates were used to ensure maximum productivity and a production of 1 mg mL-1 of colicin E9 was achieved using either LB and synthetic medium. Then, the influence of flowrates and of different growth media on the first step of purification was assessed using affinity chromatography, showing that the synthetic medium allowed higher recovery of the product in the chromatography step. Bioprocessing of the phages K and T3 was carried out in shake flasks and 5L fermenters, researching the best parameters for producing the highest titres. The effect on the final titre of a variety of multiplicities of infection (MOI), the ratio of phages out of bacteria at the moment of infection, was evaluated. For both phages the MOI that ensured the highest phage titre, 1011 PFU mL-1, was 0.01. The parameters measured were then used for continuous production of bacteriophage T3. Continuous phage T3 production was carried out using a novel reactors layout, composed by 3 separated stages. This set up allowed to divide the production of the host cells, carried out in the first reactor, from the infection, carried out in the second reactor, and from the final amplification step, carried out in the third reactor. The synthetic medium with glucose as only carbon source and the dilution rate (D, the volume of media that flows in the reactor per hour) were used to control the growth rate of the host, which strongly influenced the final production of bacteriophages. Different D iii were tested, from 0.1 to 0.6 h-1 and the independent control of the dilution rate of the first and the second reactor allowed to produce phage lysates at the same concentrations as in batch process, reaching a constant production of 2x1011 PFU mL-1 when D = 0.5 h-1 was used in the first reactor. Moreover, research on the downstream process of phage bioprocessing was conducted, aiming to reduce the host cell protein burden from phage samples using scalable techniques. The purification of bacteriophages from complex medium was performed using ultrafiltration in batch mode in a stirred cell (SC) and in using tangential flow filtration (TFF). The high shear stress produced SC decreased the viability of the tailed phage K of 2 log10 in 2 h and the positive effects of purification were cancelled by the loss of titre. Phage T3, which has a short tail, showed no side effects due to shear stress. TFF was then tested and both phage K and T3 were purified without significant viability loss. Size exclusion chromatography was used to assess the final protein concentration and showed a decrease of host cell protein load in both phages samples purified by ultrafiltration. Samples of both phages were mixed with a positively charged resin used for anion exchange chromatography and it was shown that phages that have been filtered using ultrafiltration can bind 10 times more than phages of the lysate. The main aim of purification phages produced from Gram-negative bacteria, such as T3, was to reduce the endotoxin levels (or lipopolysaccharide, LPS). Endotoxins are a major contaminant that can cause serious problems if administered and must be removed from drug products. The target was to bring the LPS levels from the initial concentration of 5x106 EU mL-1 (Endotoxin Units per mL) below the limit of 5 EU mL-1. Endotoxin removal was performed using ultrafiltration and liquid-liquid extraction (LLE). The first method tested was the TFF ultrafiltration, carried out in batch mode or in diafiltration mode. Batch ultrafiltration showed a decrease of LPS of 2 log10, either starting from a phage T3 sample from LB or from SM. Diafiltration TFF reached 3 log10 reduction of LPS in both LB or SM samples, but in LB samples it took 20 h and in SM samples only 4 h, showing how a chemically defined medium could improve the purification of phages produced from Gram-negative hosts. The other method tested to further reduce LPS concentration was liquid-liquid extraction, using 1- octanol as organic phase. Many different conditions of LLE were tested, such as length of contact time between aqueous and organic phases, different mixing methods and number of extractions. Liquid- liquid extraction using octanol ensured a reduction of endotoxins of 4 log10 from the initial concentration. This technique was then followed by ion exchange chromatography, which allowed to reach a final concentration of 102 EU mL-1. Finally, analysis on how endotoxins concentrations affected the aggregation of phage T3 were performed, showing how phage T3 aggregates in presence of endotoxins whilst it has a low polidspersivity at low endotoxins concentrations. This thesis showed how is it possible to produce bacteriophages and an antimicrobial protein at high concentrations using a continuous production mode and a chemically defined medium. The positive effects of the synthetic medium on the downstream steps were also illustrated, such as a lower burden for ion exchange columns and a faster reduction of endotoxin concentration from the samples.

Streptococcus pneumoniae (the "pneumococcus") is a major public health problem, leading to significant morbidity and mortality worldwide. The global increase in antimicrobial-resistant pneumococci is of serious concern, which necessitates investigations into novel antimicrobial strategies. Two promising areas of research are the exploitation of phages (viruses that infect bacteria) and bacteriocins (antimicrobial peptides produced by bacterial species to kill other bacteria). This thesis uses genomic approaches to address questions related to the prevalence, diversity and molecular epidemiology of bacteriocins and prophages (phage genomes that are integrated into bacterial chromosomes). The first part analysed >6,200 pneumococcal genomes and discovered 14 novel bacteriocin gene clusters. The molecular epidemiology of the bacteriocin clusters was investigated in the context of the pneumococcal population structure. The results revealed extraordinary bacteriocin diversity among pneumococci and the majority of bacteriocin clusters were also present in other streptococcal species. Genomic hotspots for the integration of different bacteriocin gene clusters were discovered. Bacteriocin genes were found to be transcriptionally active when the pneumococcus was under stress and when two different strains were co-cultured in broth. The second part describes the molecular epidemiology of satellite prophages (prophages that rely on the host and an additional helper phage for survival) in a large global and historical pneumococcal dataset. Forty-four unique satellite prophages were newly identified, which had persistent associations with specific widely-circulating pneumococcal clonal complexes over many decades. Collaborative experimental work demonstrated that one of these satellite prophages was associated with virulence in a murine model of infection. RNA sequencing revealed that satellite prophage genes were overexpressed when pneumococci were grown planktonically versus in in vitro biofilm experimental conditions. The final part analysed >1,300 genomes of 70 different Streptococcus species and identified nearly 800 prophages and satellite prophages. The data showed that prophages and satellite prophages were widely distributed among streptococci and constituted two distinct entities with little effective genetic exchange between them. Contrary to the current dogma that suggests prophages are bacterial species-specific, there was convincing evidence that transmission of prophages occurred between genetically different streptococcal species. These results broaden our understanding of bacteriocins and phages among streptococci and uncover many areas for future studies.

The increasing incidence of infections due to antibiotic resistant bacteria has led to renewed interest in bacteriophages (= phages) and phage therapy. Although phage therapy has been applied to control bacterial diseases in plants, poultry, livestock and humans, its application in aquaculture is still relatively limited. The emergence of phage-resistant bacterial mutants has been considered to be one of the major limitations of phage therapy. This study aimed to (i) isolate and characterise phages; (ii) select phages and their bacterial hosts to set up in vivo phage therapy models with aquaculture animals, and estimate the efficiency of phage therapy; (iii) investigate the generation and characteristics of phage-resistant mutants, and thus estimate the consequence of applying phage therapy when phage-resistant mutants emerge; and (iv) discuss the prospects for application of phages in aquaculture. Two Vibrio isolates and their phages were isolated from a Scottish marine fish farm. Based on the results of conventional phenotype testing and 16S rRNA gene sequencing analysis, the two vibrios, V9 and V13, were identified as Vibrio splendidus and Vibrio cyclitrophicus, respectively. The bacterial characteristics including morphology, temperature and salinity range of growth, production of extracellular enzymes, and the possession of virulence genes were examined. According to the morphological characteristics observed using transmission electron microscopy by negative staining, phage PVS9 of V. splendidus V9 was identified as a myophage, while phage PVC13 of V. cyclitrophicus V13 was identified as a siphophage. The phages could only lyse one bacterial host strain and their genomic DNA was double stranded with a size of ~46 kb. The two Vibrio isolates were found to be non- or of low virulence to rainbow trout, goldsinny wrasse and Artemia in pathogenicity experiments. Thus an in vivo phage therapy model could not be set up using these Vibrio isolates and their phages. Two phages pAS-3 and pAS-6 were isolated using the Aeromonas salmonicida subsp. salmonicida Hooke strain as the host. Phages pAS-3 and pAS-6 had a similar genome size of ~50 kb, and the same relatively narrow host range within A. salmonicida subsp. salmonicida strains. The siphophage pAS-3 formed clear plaques and inhibited A. salmonicida Hooke growth in vitro completely for at least 18 hours when using MOI = 1,000, whereas the podophage pAS-6 formed turbid plaques and weakly inhibited Hooke growth. Rainbow trout exposed by intraperitoneal injection with 0.1 mL of the raw phage preparations at a concentration of 108 PUF mL-1 showed no adverse effects over 14 days. In the phage therapy trial, fish were firstly injected with 1 x 102 CFU fish-1 of A. salmonicida Hooke, then immediately injected with phage preparations of pAS-3 and pAS-6, respectively, using MOI = 10,000. Compared with the control group (which did not receive phage treatment), phage treated groups showed a delay in the time to death, and lower mortalities. However, the mortalities and time to death between phage treated and non-treated groups were not significantly different. Phage-resistant mutants of pathogenic A. salmonicida strain Hooke were induced by repeatedly challenging with phage pAS-3. One of the mutants, termed HM, was chosen to compare the characteristics with the parental wild-type strain Hooke. Test results including the formation of ‘smooth’ colonies on TSA, autoagglutination negative, the formation of creamy colonies on Coomassie Brilliant Blue agar, and the degradation of a thick/furry layered structure on the cell surface indicated a deficiency of the A-layer in the phage-resistant mutant HM. Therefore, it was deduced that the A-layer either directly acted as the receptor of A. salmonicida phage pAS-3, or was affected indirectly by the change of an unknown phage receptor. The greater wax moth larvae model was used to compare the virulence of the phage-resistant mutant HM and the parental wild-type strain Hooke, as it is an ethically acceptable animal model, which has the advantages of being low cost and convenient for injection, and is also a recognised alternative model for bacterial pathogens of fish. The results showed that virulence of the phage-resistant mutant HM did not decline in the greater wax moth larvae model compared with that of the parental wild-type strain Hooke. In conclusion, different approaches were used to isolate and characterise phages from different aquaculture environments for potential use in phage therapy. A rainbow trout model was set up using pathogenic A. salmonicida strain Hooke and two A. salmonicida phages pAS-3 and pAS-6. The use of phage treatment led to lower cumulative mortalities and delay to the time of death, although the differences between the groups were not significant, futher work is required to determine if these phages have potential in phage therapy. The consequence of applying phage therapy when phage-resistant mutants emerge was estimated based on their characteristics and virulence, and no decline in virulence of the phage-resistant mutant from this study indicates the importance of fully testing the virulence of phage-resistant mutants before carrying out large scale field trials of phage therapy. It appears feasible to use phage therapy as an alternative approach to control bacterial infections in aquaculture, but further studies are required to focus on improving effectiveness, and also to overcome the concrete limitations and hurdles in application and commercialisation. Moreover, a broader range of applications of phages in aquaculture should be explored.

Shiga toxin (Stx) is a bacteriophage (phage)-encoded virulence factor of the Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 implicated in the pathogenesis of renal tissue damage and bloody diarrhoea in human. Cattle are the main asymptomatic reservoir for EHEC O157:H7 with the lymphoid-follicle rich areas of the terminal rectum identified as the primary colonisation site. However, the significance of Stx during bovine intestinal colonisation by EHEC O157:H7 remains unclear with mixed findings described in published studies. The objective of this study was to investigate if Stx and the Stx-encoding phage significantly contribute to EHEC O157:H7 colonisation particularly at the bovine terminal rectum. The expression of Stx receptor, Globotrioasylceramide (Gb3) at the bovine terminal rectum was analysed by fluorescence microscopy, revealing a similar pattern of Gb3 detection in the bovine colon with scattered positive detections limited to sub-epithelial, mesenchymal-associated cells. Purified Stx2 treatment of Gb3+ and Gb3- epithelial cell lines for 6 to 18 hours produced no effect on the cell cycle and proliferation. CD3+/CD8+, CD3+/γδ+ and CD21+ cells were significantly different between calves infected with EHEC O157:H7 Strain 9000 (Stx2a+/Stx2c+) and the uninfected calves, but not in calves with Strain 10671 (Stx2c+). Stx did not interfere with IFN-gamma (IFN-γ)-activation of the JAK/STAT1 pathway in epithelial cells. Bovine EHEC O157:H7 strains isolated from Scottish cattle farms in the IPRAVE study (Phage type 21/28 and 32) were used for a series of bacterial phenotypic characterisation assays. Total Stx production, Verocytotoxicity, growth in a competitive environment, epithelial cell adherence and Galleria mellonella virulence assay were performed to compare the IPRAVE EHEC O157:H7 strains (PT21/28 and PT32) and the isogenic Stx-phage mutants. Stx levels produced by the bovine-originated EHEC O157:H7 strains were significantly lower than that of the human isolated strains. The absence of Gb3 on the bovine terminal rectal epithelium, the non-significant changes in the cell cycle along with the uninterrupted IFN-γ activation of the JAK/STAT1 pathway in intestinal epithelial cells and the minute quantities of Stx generated by EHEC O157:H7 bovine strains suggest that the toxin is not involved in colonisation directly, at least at the intestinal epithelial level. Although future work is required to explain the mechanisms underlying the observed EHEC O157:H7 phenotypic changes particularly in the Stx-phage mutant strains, the work done has proven that the Stx-encoding phage indeed has the ability to exert changes in the bacterial cell leading to changes in bacterial phenotypes, which in turn, might affect the colonisation of the bovine intestine.

Haptens are small to be recognised by the immune system unless conjugated to larger carrier molecules. Characterisation of the binding sensitivity and specificity of anti-hapten antibodies, in relation to their targets, were the main focus of various previous studies. However, an understanding of the ability of antibodies to recognise haptens, at the molecular level, is still unclear. This is even factual for sheep antibodies which have generated the best anti-hapten binders. Haptens were conjugated to large carrier proteins, and were used in sheep immunisation. An antibody library was constructed, after successive rounds of antigen boosts, as representative of the extracted antibodies' genes taken from isolated sheep lymphocytes. Antibodies were selected against the hapten antigens (SQA, POR, 5BS, and HSL) using a bio-panning technique, and expressed as soluble scAb proteins. All the unique clones, that demonstrated promising recognition of the free hapten antigens, were extensively characterised. The isolated anti-SQA antibodies showed the structural naivety of SQA difficult to be recognised with high specificity and sensitivity. Although both POR and COP possess relative structural rigidity, but the polar groups of POR were necessary to generate more sensitive and specific antibodies when compared to the hydrophobic COP. Whereas the "optimum hapten structural model" was demonstrated by HSL, which has combined relative backbone firmness, with the presence of polar groups. In order to specifically understand the antibody-antigen interaction process, it was essential to investigate the effect of these haptens on the amino acid distribution, complementarity determining regions (CDRs) length, and the canonical classification of these antibodies. The canonical classification was affected by the CDR length and specific amino acids at exact positions. Sheep genetic diversity and age have influenced both the CDR length and canonical classification of the examined CDRs. In addition, the highly stringent panning protocols have necessitated the antibodies to conserve specific positions to enable the development of the required binding pockets. Anti-hapten antibodies recognition of haptens was by establishing complementary binding pockets to which the haptens can conveniently fit into and bind. These complementarities were examined in term of pockets' size, shape, and electrostatics potential. In addition, the computational docking affinity and experimental binding sensitivity can be correlated, when factors that might influence the antibody behaviour are taken into consideration, such as aggregation, adsorption, protein expression, solvent solubility and stability.

A collection of three newly isolated Salmonella bacteriophages, vB_SenS-Ent1, vB_SenS-Ent2 and vB_SenS-Ent3 was established. These bacteriophages were characterised by electron microscopy, host range, sensitivity to restriction enzymes and profiles of structural proteins on SDS-PAGE gels. The complete genome sequences of each bacteriophage were established to greater than 30x coverage and bioinformatics analysis identified the functions of a number of coding sequences. Since the last update of virus taxonomy by the ICTV a number of additional genome sequences for bacteriophages infecting the genus Salmonella have been reported in the literature. To date, all but one of the Siphoviridae comprising the Salmonella bacteriophages with fully sequenced genomes remain unclassified by the ICTV. Comparative genomic analysis reveals that a number of these phages form a coherent group within the Siphoviridae and supports the establishment of a new genus, the “Setp3likeviruses”. The proposed genus includes 5 bacteriophages infecting Salmonella; SETP3, vB_SenS-Ent1, SE2, wksl3 and SS3e, and 5 infecting Escherichia; K1G, K1H, K1ind1, K1ind2 and K1ind3. This group share identical virion morphology, have terminally redundant, circularly permuted genomes ranging between 42-45 kb in size and are characterised by high nucleotide sequence similarity, shared homologous proteins and conservation of gene order. Bioluminescent bacterial reporters, transformed to express the luxCDABE operon of Photorhabdus luminescens, were used to establish the activity of the vB_SenS-Ent bacteriophages in microtitre broth lysis assays, efficacy as biological control agents for the removal of Salmonella in contaminated foods and for spatial measurements of plaque expansion in agar overlays.