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The increase of multi-resistant bacteria highlights that the golden era of antibiotics is ending and that alternative treatmentsare urgently needed. Phages have been historically used to treat bacterial infections prior to the discovery of antibiotics and have gained renewed interest in the past decade. Despite the advantages of phage therapy over traditional antibiotic usage, a number of concerns persist over their clinical application centring on their efficacy and safety. This thesis presents four papers that focus on the isolation and characterization of phages that target reference strains and drug-resistant strains of E. coli as well as their infection dynamics and kinetics. In Paper I, six of thirty isolated phages were selected to be characterized for their growth parameters and host range using two commonly used methods. The study showed that the host range (an important selection criteria for phages) of the phages can change based on the assessment method and that the lysis efficiency of phages is host-dependent. The study suggests that standardised methods to assess the host range and lytic activity of phages are required to reduce result variability between research groups. Paper II investigated a rare phage with C3 morphotype from the Podoviridae family and characterised it via genomic, proteomic, morphologic and phylogenetic analysis. The study revealed previously unseen aspects including the formation of a honeycomb structure comprised of phage head during DNA packaging, the possible contractile nature of the tail and the 280 million year co-evolution between the major head protein and the scaffolding protein. Paper III highlights the need to take the immune system into consideration when designing phage therapeutics. In the study, four purified structurally distinct phages (selected from the three main phage families) were exposed to human cells (HT-29 and Caco-2 immortalised intestinal epithelial cell lines and donor-derived peripheral blood mononuclear cells) and the immunogenicity of the phages determined. Phage immunogenicity was shown to vary in a concentration and phage dependent manner with SU63 (a Myoviridae) being the most immunogenic phage and SU32 (a Siphoviridae) the least immunogenic. In the presence of human cells and a suitable host, phages were shown to maintain their killing efficacy as well as the ability to proliferate. Paper IV studies the infection dynamics of an experimental two-phage cocktail against a single bacterial host in vitro and in silico. However, in silico analysis and in vitro analysis produced conflicting results, in which mathematical modelling predicted the complete clearance of bacteria for all treatment scenarios whereas experimental results showed a 1-3log10 reduction in bacterial content. Practical experiments also showed increased anti-bacterial activity when the time between the additions of each phage was varied. This discrepancy suggests that the current mathematical model is unsuitable due to the inability to account for discrete variables such as interference. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p><p> </p>

The global increase in antibiotic resistance levels in bacteria is a growing concern to our society and highlights the need for alternative strategies to combat bacterial infections. Bacterial viruses (phages) are the natural predators of bacteria and are as diverse as their hosts, but our understanding of them is limited. The current levels of knowledge regarding the role that phage play in the control of bacterial populations are poor, despite the use of phage therapy as a clinical therapy in Eastern Europe. The aim of this doctoral thesis is to increase knowledge of the diversity and characteristics of bacterial viruses and to assess their potential as therapeutic agents towards multi-resistant bacteria. Paper I is the product of de novo sequencing of newly isolated phages that infect and kill multi-resistant Klebsiella pneumoniae. Based on similarities in gene arrangement, lysis cassette type and conserved RNA polymerase, the creation of a new phage genus within Autographivirinae is proposed. Paper II describes the genomic and proteomic analysis of a phage of the rare C3 morphotype, a Podoviridae phage with an elongated head that uses multi-resistant Escherichia coli as its host. Paper III describes the study of a pre-made phage cocktail against 125 clinical K. pneumoniae isolates. The phage cocktail inhibited the growth of 99 (79 %) of the bacterial isolates tested. This study also demonstrates the need for common methodologies in the scientific community to determine how to assess phages that infect multiple serotypes to avoid false positive results. Paper IV studies the effects of phage predation on bacterial virulence: phages were first allowed to prey on a clinical K. pneumoniae isolate, followed by the isolation of phage-resistant bacteria. The phage resistant bacteria were then assessed for their growth rate, biofilm production in vitro. The virulence of the phage resistant bacteria was then assessed in Galleria mellonella. In the single phage treatments, two out of four phages showed an increased virulence in the in G. mellonella, which was also linked to an increased growth rate of the phage resistant bacteria. In multi-phage treatments however, three out of five phage cocktails decreased the bacterial virulence in G. mellonella compared to an untreated control. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Methicillin-resistant Staphylococcus aureus (MRSA) is among the most important pathogens affecting the human race in our time. In spite of recent medical advances, our therapeutic choices for MRSA infections remain limited due to the propensity of this organism to develop resistance to antimicrobials. Therefore, there is a continuing need to develop newer methods of treating MRSA infections. This dissertation examines the effects of bacteriophages 88 and 92 on ten clinical isolates of MRSA from the central Florida area. . For the majority of the MRSA isolates, bacteriophages 88 and 92 were unable to induce lysis. However, bacteriophage 88 was found to lyse MRSA Sample 94. Reduced cytotoxicity and apoptosis due to MRSA Sample 94 was also observed. This protective effect was most notable in the 1:10-6 concentration of MRSA 94 and bacteriophage 88. In addition, this effect was observable with both immediate inoculation of the cell culture with the MRSA concurrent with the bacteriophage and with bacteriophage applied one hour after initial inoculation of the MRSA. This effect was likely due to the increased replication of the bacteriophage in the actively growing bacteria found in the 1:10-6 samples. The bacteria in the 1:10-6 concentration were likely more able to replicate in comparison to the higher bacterial concentrations because of less competition between the bacteria for the limited nutrients in the 1:10-6 concentration. The long-term goal of this study is the development of a bacteriophage-containing ointment for the control of MRSA nasal carriage. In addition, the concept of bacteriophage therapy may open a new horizon in controlling infections such as those caused by MRSA. Finally, as for future studies, it would be informative to be able compare these results with other MRSA isolates and bacteriophages samples to examine the effects across a wider sample of bacteria and bacteriophages. In addition, it would be interesting to examine the possibility of being able to modify the bacteriophage in order to allow lysis of the previously resistant bacterial strains.