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As the threat of antibiotic resistance increases, it is now more important than ever to focus on the development of new strategies to combat pathogenic bacteria. Since the cell membrane is the gateway to a bacterial cell, it is crucial that interactions occurring here are fully understood. This thesis focuses on three areas of research which, when brought together, could allow us to monitor these membrane systems at an unprecedented level of precision. The first is the development of the supported lipid bilayer model that represents bacterial cell membranes in an *in vitro* setting. These models have existed for some years, but recently a breakthrough has been achieved where supported lipid bilayers can now be created which contain naturally occurring components of cell membranes, thus making them more biologically relevant. The second area is high resolution microscopy techniques which can image these bilayer systems at an unprecedented level of resolution, thus shedding new light on the interactions occurring here. The third focus is the emerging field of bioelectronics. Bioelectronics exists at the junction of biology, chemistry and materials science; it aims to combine advances in these research disciplines in order to integrate biological systems with electronics and therefore monitor biological processes electrically. By combining bioelectronics with supported lipid bilayer models, the aim is to create a quantitative, rapid and label-free readout for biological processes occurring in the membrane. The thesis begins by using the simplest types of supported lipid bilayer models to show how a combination of microscopy and bioelectronic techniques are used to measure these systems. A membrane disrupting antibiotic is used for proof-of-concept studies to show how bioelectronics can monitor these disruption effects. A membrane protein transporter is then incorporated into these bilayer systems. Microscopy and bioelectronic techniques are combined with biochemical assays to provide a multiparametric picture of protein functioning in various conditions, for instance in the presence of an inhibitor compound, therefore showing the platform's application in antimicrobial development. The complexity of the bilayer models is then increased substantially, using vesicles derived from bacterial cells to create bilayers which contain naturally occurring components of the cell membrane. The strength of the cutting-edge microscopy techniques is displayed here, imaging interactions occurring at the surface of these membranes at a single molecule level of resolution. These complex bilayer systems are also demonstrated to act as a screening platform for bacteriophage interactions - viruses that specifically interact bacteria and therefore have long been touted as an alternative to conventional antimicrobial strategies. It is hoped that the platform developed here may help in identifying bacteriophage against pathogenic bacteria of interest. Finally, the versatility of supported lipid bilayers is explored using vesicles derived from a wider range of bacteria. This shows that the general monitoring platform developed in this thesis can be applied to a variety of bacterial systems. In conclusion, the work presented here focuses on the development of bilayer models that faithfully represent bacterial membranes, and crucially the monitoring of bilayer functions using a combination of bioelectronic readouts and high-resolution microscopic imaging.

Bacteriophages, viruses that infect bacteria, are the most abundant biological entities on earth and one of the most potent evolution-driving forces in nature due to their capacity to facilitate horizontal gene transfer. Tailed bacteriophages have been studied intensively, and they rely on tail spikes or tail fibres to recognise their cognate host receptors when adsorbing to bacteria. This adsorption step is crucial for the infection process; correspondingly, it is also a key factor dictating host range specificity. Viunalikeviruses are lytic-tailed phages with dsDNA genomes of ~150 kbp that exhibit extensive sequence homologies within the family and generalised transduction capacity. The Viunalikeviruses are virulent phages, and representative examples have been isolated on Gram-negative bacteria, including animal and plant pathogens such as Salmonella, Escherichia, Klebsiella, Shigella and Dickeya. Although there has been limited analysis of Viunalikevirus receptors, the primary receptor of the defining example of this viral group (the Vi01 phage) in Salmonella Typhi has been identified as the Vi capsular polysaccharide (CPS). This study aimed to develop a promiscuous phage receptor expression system allowing infection in a broad range of heterologous hosts. The S. Typhi Vi CPS biosynthetic operon was cloned into a broad host range cosmid and horizontally transferred into a diverse group of Gram-negative species. The expression of the Vi CPS was correlated with the infection of ΦVi01 on all the evaluated species. One of the highlights of this study is the artificial host range extension of ΦVi01 into 19 different species belonging to three different taxonomic Orders. Additionally, the effect of different bacterial defence mechanisms on ΦVi01 virulence was studied. ΦVi01-mediated transduction was observed in most of the analysed hosts, allowing horizontal gene transfer between distantly related hosts. Also, strains expressing the Vi CPS were used to isolate environmental phages recognising this phage receptor in different heterologous hosts. Two new Vi CPS-dependent phages, ΦTKR01 and ΦTKR02, were identified and characterised. This study results are the first description of a synthetic heterologous promiscuous system for phage host range extension. This simple system could have potential applications for genetically engineering non-canonical bacterial species. Additionally, the heterologous expression of this phage receptor serves as a tool for isolating environmental phages capable of recognising this structure.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Paratuberculosis (Johne's disease) in mammals. This is a chronic, untreatable and ultimately fatal wasting disease that primarily affects ruminants. There are also some concerns about the zoonotic potential of MAP, as MAP infection has been linked to Crohn's disease and various other autoimmune disorders. Many countries have introduced MAP control programmes, both to improve the health of their livestock and to limit human exposure to MAP. However, these programmes have had varied success, in part due to the lack of sensitive and specific MAP detection methods. Typically, MAP is detected by ELISA, PCR or culture, but none of these tests are completely sensitive and specific for MAP. Therefore, the aim of this PhD research was to investigate the biology of MAP in order to improve current MAP detection methods. Particular emphasis was placed on MAP culture, as this is the current 'gold standard' detection method for viable MAP. This means that novel MAP detection methods must be validated against culture, despite its lack of sensitivity. Other areas investigated included: (1) using MAP-specific monoclonal antibody and peptide binders to identify epitopes of MAP antigens with the potential to be used for the development of immune-based MAP diagnostic detection assays, and (2) characterisation of recently isolated mycobacteriophages with potential to be used in place of mycobacteriophage D29 in bacteriophage-based detection methods for MAP. MAP may be cultured in liquid or solid media. Culture in broth is faster, but culture on agar is arguably more useful as single colonies are obtained which can be isolated and stored for further studies. For this reason, two novel MAP solid culture methods were developed during this study that have the potential to reduce the time until MAP colonies become visible on agar. The first was an optimised solid culture medium consisting of Middlebrook 7H9 broth with 1.0% Tween 80, 0.019% casitone, 1.4% bacteriological agar, 10% egg yolk, 10% ADC and 1.65 µg/ml Mycobactin J. MAP growth became visible two weeks earlier on this new QUB medium than on Herrold's Egg Yolk Medium (HEYM). The second novel culture method was designed to take advantage of speed of culture in broth while still enabling single colonies to be obtained. The '2-in-1' system consists of a tissue culture flask or universal tube containing a layer of agar along with a small volume of broth, enabling samples to be enriched in broth prior to inoculation of the agar surface. The '2-in-1' culture system significantly improved the incubation periods and final colony numbers of MAP cultured on HEYM compared to culture on solid media without an enrichment period in broth. A novel antimicrobial cocktail was also developed to reduce contamination of cultures when isolating MAP from raw milk samples. The new cocktail consisted of 8 µg ampicillin, 16 µg nalidixic acid and 50 µg amphotericin B per ml medium. The new cocktail inhibited non-target microorganisms from raw milk samples more effectively than the standard 2.5% PANTA but was also detrimental to MAP viability. Further optimisation will be required before it could be used for MAP culture. With a view to potentially being able to replace MAP whole cell antigens used in current ELISA assays with peptide mimics of key MAP cell surface epitopes, during Covid lockdown time was spent on computational analysis of several 12-mer peptide mimics identified previously through phage display biopanning of the MAP-specific antibodies MAb 6G11 and MAb 15D10, and a 12-mer peptide Biotin-EEA402. The most promising peptides identified were peptide sequence TAKYLPMRPGPL for MAb 6G11, peptide sequence HNDNNTDQTWWW for MAb 15D10, and peptide sequence VYSPCGPCRRFL for BiotinEEA402. However, there was evidence that the peptides may bind to multiple Mycobacterium spp. rather than MAP specifically. These results will need to be confirmed experimentally with ELISAs. Finally, because phage-based MAP detection methods show promise for detecting viable MAP quickly, four recently isolated mycobacteriophages were characterised to determine if they might substitute or complement the currently employed D29 mycobacteriophage. Phage DNA sequencing and subsequent bioinformatics indicated that all four isolates were identical members of the H1 subcluster of lytic Siphoviridae phages. On the basis of the limited amount of laboratory work possible, the ability of the novel phage isolate to infect and amplify within MAP is suspected, but has yet to be conclusively demonstrated.

Bacteriophage (phage) are viruses which infect and replicate within bacteria cells. Phages replicate by finding a host bacteria cell, adsorbing to it, injecting their DNA, hijacking the host machinery to produce more phage, and finally lysing the cell to release the new phage. The evolutionary dynamics of phage populations are therefore strongly tied to their interactions with the host bacteria. In a spatial context, I was able to show that unavoidable physical interactions between the virus and host hinder viral dispersal, leading to a phage population in which stochastic fluctuations are much weaker. This allows beneficial mutations to establish more easily and deleterious ones to be purged, making the phage population more adaptable to changes in the environment. To facilitate better comparisons between theory and experiment in spatial contexts, I also developed a variety of new experimental techniques. These were aimed at addressing shortcomings of existing techniques, which are almost exclusively based on population averages and are carried out in well-mixed liquid cultures, making their applicability to spatial settings challenging. Finally, phages are also able to interact with each other via their host, by encoding superinfection-exclusion mechanisms which, after initial infection, prevent subsequent phage from successfully infecting that host. Using stochastic simulations, I show that in the long term such mechanisms limit the adaptive potential of the phage, by limiting the genetic diversity in the population and reducing the efficiency of selection. In the short term however, having such a mechanism provides a very large advantage over other phage in the population which lack such a mechanism, possibly explaining the ubiquity of such mechanisms in nature.

The outbreak of COVID-19 demonstrated the depth of global pandemic and the extent to which it can affect various aspects of human life, from direct health issues to indirect economic and social damages. Antimicrobial resistance, one of the world's most health threatening issues, could have very similar effects. Phage Therapy has been demonstrated as a possible alternative to antibiotics in the former Soviet Union but has only recently gained attention elsewhere. Several factors are preventing the fast commercialisation of bacteriophages as an alternative to antibiotics. This work addressed two of these issues through a series of studies, the stability and drug dosage form. Spray drying bacteriophage, Salmonella Felix O1, using a 2:1 ratio of trehalose and Eudragit polymers (S100 and L100) at an outlet temperature of 80°C, significantly improved phage stability during the encapsulation process, storage, and once exposed to the acidic environment. Tableting of encapsulated phages using the direct compression tableting method was achieved without damaging the encapsulated phages. It also further improved acid stability and provided additional protection during storage under 65% relative humidity (RH). Phage viability and tablet quality are both dependent on the excipients used for encapsulation and those used before tableting. The addition of 2% Magnesium Stearate (Mg-St) reduced friability and improved phage viability during the storage compared with those with lower content. Tableting of PS21-S100 powders was possible using a maximum of 15 kN compaction pressure, without damaging the phage while for PS21-L100 no damage was observed up to 20 kN. The disintegration of tablets each weighing 290 mg with a diameter of 10 mm was affected both by the compaction pressure. The disintegration of tablets was reduced with an increase in compaction pressure. Also, the excipients influence the disintegration with PS21-S100 experiencing lower mass loss in comparison with PS21-L100. In both formulations, friability was modified by increasing compaction pressure from 5 kN to 10 kN, acid protection was possible even at pH 1 and no batch-to-batch variation was observed in powders and tablets. Storage of powder and tablets indicated low temperature and humidity conditions are most suitable for storing encapsulated phages for the long term. Samples stored under 4°C and 0% RH and 20°C (uncontrolled RH) experienced one log drop over six months while those stored at 30°C and 65% RH underwent three log drops for the same period. Storage of samples at 30°C and 0% RH resulted in improved stability and demonstrated the adverse effect of moisture absorption on phage viability. The investigation into moisture content revealed moisture absorption occurred in samples stored at 20°C and 30°C and 65% RH, explaining the decline in phage viability under these conditions. Finally, drying of samples using vacuum drying without damaging the phages to 3% moisture content was possible but did not result in improved stability at 30°C + 0% RH. From these studies, it can be concluded that the current formulation and encapsulation methods can improve phage viability and acid protection and enhance long term storage. Also, tableting provides additional protection in extreme conditions of acidic and very humid environment. Finally, PS21-L100 samples performance was better during long term storage suggesting the possibility of eliminating the cold supply chain.

The COVID-19 pandemic has demonstrated the impact that untreatable infectious pathogens can have on society but an existing threat, if unaddressed could be even more devastating. Excessive deaths from infectious diseases were thought to be a thing of the past but antimicrobial resistance could cause the 700,000 annual deaths reported from resistant organisms to rise. New or alternative treatments to target Gram-negative pathogens are urgently required, alongside a greater understanding of the mechanisms of resistance to currently available therapeutics. Here I aim to advance the field of antimicrobial research through the exploration of the potential alternative therapy, bacteriophages, as well as investigating novel ways of cultivating previously unidentified antibiotic producing microorganisms. I demonstrate through novel microfluidic and single cell technologies (previously underutilised for phage assessments) that strategies for phage resistance in Escherichia coli are environment - structure dependent. In more complex environments, bacteria favour phenotypic over genetic resistance, which occurs through a diverse range of mechanisms such as filamentation or reduced receptor expression, and that extensive heterogeneity exists within the population. Such findings are important to enable the evolutionary and ecological dynamics of bacteria-phage interactions to be predicted and manipulated if they are to become a viable therapy in the clinic. I further show that using these microfluidic systems, the environment where phage-bacteria interactions occur can be tightly moderated and manipulated. Exposure duration, nutrient availability and even bacterial growth phase can be altered to optimise killing efficacy and observe the different single cell phenotypes of both surviving and susceptible cells that occur in response to phage. New treatments to target potential biothreat agents are also required. Here I have shown novel treatment options for Burkholderia involving a recently environmentally isolated phage in combination with existing antibiotics. Through antibiotic-antibiotic and phage - antibiotic combinations, I have been able to demonstrate improved clearance of B. thailandensis populations in vitro whilst lowering the required concentration of antibiotic. Although studies involving bacteriophage as an antimicrobial therapy are proving promising, they have not replaced the urgent need for new antibiotics. The majority of our current antibiotics are derived from environmental bacteria, but no new compounds have been licensed for decades. Here culturing platforms were designed to facilitate the growth of previously unculturable bacteria in an estuarine mud environment in situ. This method is still low-through put and additional parallel approaches are required such as microdroplet/ microfluidic systems or culture independent approaches like metagenomics. Ultimately, the future of antibiotic discovery to target the antibiotic resistance crisis lies in combined, parallel investigations utilising all available knowledge and resources in a unified approach for the treatment of infectious diseases.

Due to the limitation of fossil fuels supply and the increased concern for carbon emissions which results in global warming, fuel cell has been rapidly developed and is widely regarded as an attractive power supply device which generates electrical energy from the chemical energy of a fuel directly. Electrocatalytic oxidation reaction of the fuel directly harvests chemical energy with significant higher efficiency at a much lower operating temperature. Direct alcohol fuel cells (DAFCs), one of the fast developed fuel cells, are a clean and renewable source of energy making them an ideal a replacement for the traditional fossil fuels. Alcohol is an attractive fuel because it is a stable liquid at room temperature, which makes it easier for storage and transportation. In this thesis, bare Pd electrocatalyst, supported by glassy carbon (GC), towards alcohol (ethanol and 1-butanol) electrooxidation reaction in alkaline media at different temperatures were firstly studied. Then a series of Pd-based electrocatalysts were developed to improve the activity and stability for ethanol electrooxidation reaction (EOR) and 1-butanol electrooxidation reaction (BOR) in alkaline media. The kinetics of alcohol oxidation reaction on Pd and Pd-based electrocatalysts were investigated and compared based on the activation energy (Ea) calculated by Arrhenius plots using the data obtained from cyclic voltammetry (CV) and chronoamperometry (CA) studies at various temperatures. When only Pd/GC electrocatalyst was tested for alcohol electrooxidation reaction within ethanol and 1-butanol, the catalysts activities and kinetics were enhanced at higher cell temperature and the peak potentials also positive shifted to higher potential while temperature increased. Ethanol had a higher positive peak current density at all temperatures while 1-butanol had a lower activation energy. The results showed that the reaction activities increased while the activation energy decreased as the length of the carbon chain of the primary alcohols increased. Then a series of nanoparticle sized Pd-based electrocatalysts were synthesized using Cu, Bi, Nb and BiNi nanoparticles supported on GC to test in the ethanol and 1-butanol oxidation reactions. The results showed that 34% Cu achieved highest current density and lowest activation energy (Ea) for EOR and 48% Cu showed lowest Ea in BOR. In case of Bi, 43% was the optimum coverage for EOR, and in BOR, 42% Bi had lowest Ea while peak current density reached highest at 50%. For the Nb study, it was shown that the best coverage for EOR was 50% and 45% for BOR. The best coverage of BiNi was 43% in EOR, which was the same with Bi, indicating both are very promising co-catalysts for EOR. The production of T4 and M13 bacteriophage were successful, both in small and large scale, supported by clearly accountable points in plaque assay and phage culture curves and best operation temperature of 37°C was determined. The initial bio-catalyst test of Phage-Cu-Pd/GC was conducted and a promising result in terms of activation energy, with even lower Ea value compared to no phage added, was achieved at 36% Cu overage.

The hypothesis of this thesis is that phages isolated from the environment can play a role in the future to reduce outbreaks of the disease listeriosis by applying phages as biocontrol agents against the pathogen L. monocytogenes and by incorporating phages into systems for the detection of this pathogen. Phage vB_LmoH_P61, a lytic phage isolated from grass silage, was applied to food matrices for the biocontrol of L. monocytogenes which resulted in the inhibition of L. monocytogenes in artificially inoculated pasteurized milk and baby spinach. The key findings from this study show the importance of the food matrix, multiplicity of infection, the growth stage of the host bacterial cells and storage temperature for the successful application of phages as biocontrol agents. Chapter 3 of this thesis also aimed to exploit phages (phage lysins) as biocontrol agents. The key findings showed that polyhydroxyalkanoate bionanoparticles displaying the full-length endolysin of phage vB_LmoS_293 or the amidase domain of this lysin, lysed and inhibited the growth of L. monocytogenes strain 473 at 37 ºC and 22 ºC. Phages were also exploited for the capture and potential detection of L. monocytogenes (Chapter 4). A recombinant receptor binding protein from phage vB_LmoS_293 was produced and shown to capture its host-bacterial cells. Three phages-vB_LmoS_C996, vB_LmoS_P11 and vB_LmoS_P7-were isolated from grass silage and characterized. The endolysins and putative receptor binding proteins were identified in the genomes of these phages, and these proteins may be exploited in future applications for the biocontrol and detection of L. monocytogenes. In this thesis, new phages have been isolated, new protocols developed for the inhibition of L. monocytogenes, a potential new delivery system of phage lysins has been shown and new protocols developed for the production and purification of recombinant phage proteins.

Bacteriophages (or phages; viruses that infect bacteria and archaea) profoundly influence microbial communities. Given the impact of the gut microbiome composition and function on human health, there is a growing focus on phages that inhabit the gut ecosystem. However, the extent of viral diversity, biology, and worldwide epidemiology of gut phages remain largely unknown. In this thesis, I carry out a comprehensive genomic analysis of gut phages by harnessing the biggest collection of phage genomes, gut bacteria isolates, and human gut metagenomes. I begin by introducing the Gut Phage Database (GPD) which is the largest genomic resource to date of human gut phage genomes and product of mining 28,060 faecal metagenomes and 2898 gut bacteria isolate genomes. I use machine learning to improve the quality of the predictions and investigate ways to organise the viral diversity in order to improve the characterisation of gut phages in downstream analyses. Afterwards, I describe common functions and auxiliary metabolic genes encoded by human gut phages. I also highlight instances of hypervariable domains which may indicate the presence of phage receptor binding proteins. I then shift the focus to the analysis of two clades of gut phages, namely the Gubaphage and the Picovirinae subfamily. The Gubaphage is a novel phage clade uncovered in this work which is highly prevalent across the world. The Picovirinae clade was the most common predicted phage taxonomy in GPD. Host assignment allows me to study patterns of phage diversity across bacterial clades of the human gut and investigate their host range. Finally, I analyse global patterns of the human gut phageome and its association with lifestyle and bacterial composition. I assess the idea of a core virome as well as in what degree my data agrees with this concept.

Since the discovery of antibiotics, there has been a revolution in treating microbial infections. However, with their increasing use, some microbes have developed a form of resistance to many of the available antimicrobial agents. Untreatable infections because of antimicrobial resistance (AMR) have recently become a global threat and it is estimated that by 2050, more than 10 million people will die per annum from resistant bacterial infections. Therefore, novel methods are needed to kill resistant microbes. One such method that may be used to kill resistant bacteria are bacteriophages (phages), viruses that infect microorganisms and cause them to lyse.

Abortive infection (Abi) is an anti-phage mechanism in which a bacterium initiates its own death upon phage infection. This prevents or decreases the production of phage progeny and protects clonal siblings in the bacterial population by an act akin to an "altruistic suicide." Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa, first found in Pectobacterium atrosepticum. ToxINPa consists of an endoribonuclease toxin and RNA antitoxin and is inactive as a heterohexameric complex until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa-mediated Abi and what mechanisms are involved. This study aimed to address this issue by first introducing ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S39006). A novel environmental S39006 phage, ΦCBH8, that is sensitive to ToxINPa-mediated Abi was isolated, characterized, and compared to its spontaneous "escape" mutants that were insensitive to ToxINPa. Characterization of ΦCBH8 led to the discovery of a new genus of T4-family phages that are all ToxINPa-sensitive. Genomic comparison of ΦCBH8 with its spontaneous ToxINPa-escape mutants led to the discovery of two distinct genetic loci that are candidates for activation of ToxINPa-mediated Abi. One such locus was a multi-gene region of the phage genome, the deletion of which enabled some ΦCBH8 mutants to escape ToxINPa. Different ToxINPa-escape mutants had variable deletions in their genomes, but this multi-gene locus was deleted in all of the deletion mutants. Experiments showed that at least two genes in this locus must be simultaneously involved in activating ToxINPa-mediated Abi and one phage gene product within the locus (ORF18) was extremely toxic to the bacterial host. Another locus was the asiA gene. This study showed that ΦCBH8 mutants with mutated versions of asiA became insensitive to ToxINPa but genetic complementation restored their sensitivity. AsiA is involved in σ-appropriation but the mutated AsiA in ΦCBH8 mutants was no longer functional. Further experiments showed that AsiA from wild type ΦCBH8 reduced ToxINPa promoter activity while mutant AsiA did not, suggesting that AsiA could be involved in activating ToxINPa-mediated Abi by perturbing toxINPa transcription. Finally, an outer membrane protein (OmpW) was defined as the ΦCBH8 receptor in S39006. Transfer of the S39006 ompW gene to other bacterial species rendered some of them sensitive to the phage thereby enabling expansion of ΦCBH8 host range. Results showed that ΦCBH8 was insensitive to ToxINPa in E.coli, suggesting that activation of ToxINPa-mediated Abi is host-dependent. The function of OmpW was explored to try to link bacterial host physiology with phage susceptibility In conclusion, this work characterized novel phage genes involved in activating ToxINPa-mediated Abi. This provided evidence that viral gene lethality and σ-appropriation might be related to the mechanism(s) for phage activation of ToxINPa-mediated Abi.

Phytopathogenic Dickeya species inflict large economic losses on a variety of crops. A lack of effective chemical control methods has generated interest in the use of bacteriophages (phages) as a novel tool for biocontrol. In the last decade, six phages have been isolated in Belgium and Poland using Dickeya solani as the host. Previous work in this laboratory has isolated ninety phages capable of infecting D. solani. The majority have been morphologically classified as members of the Ackermannviridae family. In agreement with findings in Salmonella and Klebsiella species, the capsule of D. solani is a likely receptor of Ackermannviridae family phages. Analysis of D. solani strains carrying reporter fusions suggested that the capsule genes are expressed in response to nutritional stress, however disruption of the capsular polysaccharide cluster did not significantly impact virulence. Experiments assessing capsular polysaccharide as a putative receptor for Ackermannviridae family phages in nosocomial pathogen Serratia produced inconclusive results. Phageresistance due to random transposon mutagenesis identified genes encoding transcription factors and regulators, but none directly linked to capsular polysaccharide production. Thirteen phages were capable of infecting a wider host range of Dickeya species. Morphological and genomic analysis showed that six were Podoviridae family members, whilst the other seven were Myoviridae family members. These are part of the recently defined 'hairy Myoviridae', characterised by a distinct morphology. Another member of this grouping was isolated during this study, but is more closely related to phages of Erwinia amylovora. A subset of the Ackermannviridae family phages were shown to be capable of facilitating transduction. This makes them unsuitable for use in the environment due to the risk of deleterious horizontal gene transfer. This is also true for the Myoviridae family members, but not for one of the Podoviridae family members. This phage could therefore be a promising candidate for therapeutic use.

Enteric pathogens form a large part of infectious diseases which contribute to a bulk of the healthcare costs. Enteric infections are usually contracted via the faecal-oral route or through contact with contaminated surfaces. Treatment by antibiotics is becoming increasingly ineffective due to the growing number of antibiotic resistant strains. Anti-microbial resistance poses a serious threat to the future of healthcare worldwide and necessitates the search for alternate forms of therapy. Bacteriophages (phages), are viruses which specifically infect and lyse bacteria. To introduce phages as a viable form of therapy, route of administration needs to be considered carefully. Model phages with broad host ranges are ideal for therapy however oral delivery to the lower gastro-intestinal (GI) poses several challenges. The acidic stomach environment can be detrimental to phages, rendering them inactive during passage. To overcome this challenge and improve the stability of phage during encapsulation and storage, this PhD research has been conducted. pH responsive polymers, Eudragit and alginate were used to develop composite microparticles which protected phage from acidic pH (pH 1-3). A novel method of acidifying oil was developed for crosslinking droplets in vitro to avoid the use of harsh solvent systems that can cause phage inactivation. Platform microfluidic technology was employed for phage encapsulation for the first time. Monodispersed droplets and particles were produced, offering fine-tuning of droplet diameter to tailor the release and pH protection of encapsulated phage. Process scale-up was attempted using membrane emulsification (ME) to produce larger volumes of encapsulated phage. In vitro and in-situ models investigated the efficacy of encapsulated phage-bacterial killing. Industrial scale method of spray drying, and electrospinning were also used to demonstrate the versatility of the formulation. Tableting dry powder phage, showed an effective method for producing solid dosage forms for therapy. Additionally, electrospun phage fibres also showed the potential use of pH responsive formulations in addressing wound infections. Improvement in encapsulated phage storage stability was observed with the addition of trehalose in the formulation. This research underpins the need for testing phage encapsulation for site-specific delivery and offers insight into the potential use of commercially available technologies.

A prevalent morphology in the microscopic world of artificial microswimmers, bacteria and viruses is that of a helix. The intriguingly different physics at play at the small scale level make it necessary for bacteria to employ swimming strategies different from our everyday experience, such as the rotation of a helical filament. Bio-inspired microswimmers that mimic bacterial locomotion achieve propulsion at the microscale level using magnetically actuated, rotating helical filaments. A promising application of these artificial microswimmers is in non-invasive medicine, for drug delivery to tumours or microsurgery. Two crucial features need to be addressed in the design of microswimmers. First, the ability to selectively control large ensembles and second, the adaptivity to move through complex conduit geometries, such as the constrictions and curves of the tortuous tumour microvasculature. In this dissertation, a mechanics-based selective control mechanism for magnetic microswimmers is proposed, and a model and simulation of an elastic helix passing through a constricted microchannel are developed. Thereafter, a theoretical framework is developed for the propulsion by stiff elastic filaments in viscous fluids. In order to address this fluid-structure problem, a pertubative, asymptotic, elastohydrodynamic approach is used to characterise the deformation that arises from and in turn affects the motion. This framework is applied to the helical filaments of bacteria and magnetically actuated microswimmers. The dissertation then turns to the sub-bacterial scale of bacteriophage viruses, 'phages' for short, that infect bacteria by ejecting their genetic material and replicating inside their host. The valuable insight that phages can offer in our fight against pathogenic bacteria and the possibility of phage therapy as an alternative to antibiotics, are of paramount importance to tackle antibiotics resistance. In contrast to typical phages, flagellotropic phages first attach to bacterial flagella, and have the striking ability to reach the cell body for infection, despite their lack of independent motion. The last part of the dissertation develops the first theoretical model for the nut-and-bolt mechanism (proposed by Berg and Anderson in 1973). A nut being rotated will move along a bolt. Similarly, a phage wraps itself around a flagellum possessing helical grooves, and exploits the rotation of the flagellum in order to passively travel along and towards the cell body, according to this mechanism. The predictions from the model agree with experimental observations with respect to directionality, speed and the requirements for succesful translocation.

Catheter associated urinary tract infection seriously complicates the care of an already vulnerable patient set and has been estimated to cost the UK National Health Service in excess of one billion pounds per annum. Approximately 50 % of patients catheterised for more than 28 days will experience catheter blockage due to the formation of crystalline biofilm on the eye holes, balloon and lumen of the catheter(Getliffe, 1994) as a result of colonisation by Proteus mirabilis. Blockage can lead to significant complications such as pyelonephritis and septicaemia. To date, strategies to reduce or prevent these infections from occurring have met with limited success. One potential approach to prevent catheter colonisation and blockage is the application of bacteriophages as a catheter coating. Natural parasites of bacteria, bacteriophages offer several advantages over conventional antimicrobial treatment including replication at the site of infection, specificity and, in some cases, biofilm degrading ability. Three novel bacteriophages vB_PmiS_NSM6, vB_PmiP_#3 and vB_PmiM_D3 were isolated from environmental sources and characterised phenotypically and genetically utilising electron microscopy, host range analysis and, for phages vB_PmiS_NSM6 and vB_PmiP_#3, genome sequencing via hybrid assembly. The isolated phages belong to the Caudovirales order and sequence data analysis indicated that they were lysogenic. They possess the characteristic modular architecture of their dsDNA genomes that are densely packed with coding sequence. Both phages displayed terminal redundancy which is indicative of a headful packaging strategy and both appear to be circularly permuted. Putative function was obtained for 63 % of the coding sequences for phage vB_PmiS_NSM6 and 52 % of genes identified in phage vB_PmiP_#3. The effect of these phages, either individually or as a cocktail, on P. mirabilis colonisation of urinary catheters in an in vitro bladder model was investigated. Models were run for 24 h and adhered bacteria used as an indicator of phage activity. A reduction of greater than 3 log10 was observed for phage vB_PmiS_NSM6 treated catheters in comparison to untreated controls across all three sections of catheter analysed. Phage vB_PmiP_#3 reduced bacterial adherence by 1 log10 across all sections and a similar reduction was observed with phage vB_PmiM_D3 of greater than 1 log10. These data were confirmed with scanning electron microscopy (SEM) which showed a significant reduction in crystalline deposits on the phage treated catheters. The time taken for the mineralised biofilm to occlude the catheter lumen in the presence of bacteriophages was also investigated. Time to blockage was extended by 61.49 %, 25.67 % and 52.31 % for phages vBPmiS_NSM6, vB_PmiP_#3 and vB_PmiM_D3, respectively, in comparison to controls. Phages vB_PmiS_NSM6 and vB_PmiM_D3 displayed activity on each other’s isolating strain. This enabled the assessment of a two phage cocktail. The cocktail increased time to blockage by approximately 7 % compared to single phage treatment for both bacterial isolates. These data provide some evidence of efficacy of bacteriophage pre-treatment of urinary catheters in an in vitro model of P. mirabilis infection of the catheterised bladder, despite the lysogenic nature of the phages investigated. As such, this suggests phage treatment of catheters warrants further investigation.

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.

The production of biofuels and in particular bioethanol has increased rapidly since the early 1990’s. The advantages of biofuels include reduced CO2 production, a decrease in fuel importation for many nations (notably the US and Brazil), and comparatively simple blending with fossil fuels. The production of basic fuel ethanol (1st generation) involves the use of an energy crop feedstock (corn in US and sugar cane in Brazil). The feedstock is processed via simple mechanical methods to release the simple carbohydrates, mixed with water and fermented anaerobically via S. cerevisiae yeast into ethanol and CO2. Due to the low market value of fuel ethanol, profit margins are restrictive, and as a result sterilisation and aseptic techniques are not economically viable, and contamination by environmental organisms is commonplace. The current system of biocontrol involves the addition of antibiotics, primarily penicillin and virginiamycin, to the fermentation. While these antibiotics are broad spectrum and highly effective in reducing the impact of contamination, the negative environmental impacts of antibiotic usage are well known. In order to reduce the impact of contamination and reduce reliance on antibiotics an alternative system of biocontrol is required. In this thesis various biocontrol agents are assessed, including bacteriophage, hop acids, chitosan, onion oil extract, copper and silver ions. The effect of these agents on the growth of various contaminant bacteria and a strain of S. cerevisiae is assessed and fermentations are carried out under sterile and controlled contaminated conditions to generate data on the effect of the contaminant and the various methods of biocontrol. Other possibilities investigated include the insertion of plasmids containing heat shock proteins into S. cerevisiae to enhance thermo-tolerance.

Psoroptes ovis mites are the causative parasites of sheep scab disease. It is a contagious disease which causes intense pruritus, wool loss and the development of lesions. These lesions are exacerbated by secondary bacterial infections. Bacteria appear to play an integrated role in the pathogenicity of this disease and are found in the internal cavities of P. ovis. The aim of this study was to investigate these bacterial associations, with the aim of identifying a microbial target for sheep scab control. The microbial communities associated with sheep scab were investigated using both molecular and bacteriological techniques. Several environmental niches were targeted: scab-infected fleece, internal mite cavity and excreted faecal trails. Microbial communities were very complex, with a variety of species and bacterial groups identified. Some bacteria were common to all environments, whereas others were isolated from one sample. Both natural and in vivo cultured mites were investigated in an attempt to identify universal and potentially beneficial bacteria. In addition, P. ovis mites were screened using PCR to detect potential endosymbiotic bacteria. Positive identification was made of Comamonas sp. in both natural and in vivo cultured mites; this species has been identified as an endosymbiont in other arthropods and its role in P. ovis requires further investigation. In vitro feeding experiments were carried out with P. ovis mites in the laboratory. Initially mite chambers were constructed and optimised to encourage maintenance of P. ovis off-host. A number of diets were tested and antibiotics were compared for their effect on bacteria within P. ovis. In vitro experiments revealed that P. ovis survival was significantly reduced with the administration of antibiotics and there was also evidence that they altered internal bacterial densities. The potential of bacteriophage therapy for the microbial control of bacteria associated with P. ovis was investigated. Bacteria isolated from P. ovis faecal trails were used to isolate bacteriophage from environmental samples. Sixteen bacteriophage were successfully isolated, which were infective against three mite faecal bacteria. Isolated bacteriophage were characterised by a number of methods including their response to chemicals, enzyme and infection dynamics in both solid and liquid phases. In vitro experiments with bacteriophage were also investigated, resulting in a significantly reduced mite lifespan seen with some bacteriophage lysates. The potential for using bacteriophage for the control of P. ovis mites is discussed.