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The escalating threat of antimicrobial resistance has intensified the exploration of alternative treatments, with bacteriophage (phage) therapy emerging as a potential substitute for antibiotics. While strictly lytic phages rapidly kill bacteria, temperate phages can also go dormant in their hosts. Accordingly, despite their prevalence, they are considered unsuitable for therapy. My systematic review of phage therapy in urinary tract infections (UTIs) highlighted this. This review motivated me to explore how the potential of these phages could be leveraged. Chapter 3 introduces a novel strategy to do so, exploring whether the fluoroquinolone antibiotic ciprofloxacin could synergize with temperate phages. This innovative strategy exploits the ability of the antibiotic to awaken dormant temperate phages, driving a potent synergy (≥8 log reduction) able to result in bacterial eradication. This is a potential breakthrough in the use of phages. Chapter 4 expands on this finding, establishing that a synergy exists across various drug classes with diverse mechanisms of action. Surprisingly, the synergy extends beyond antibiotics triggering the bacterial SOS-response known to wake temperate phages and also includes protein synthesis inhibitors, offering a new approach to influence the phage lysis-lysogeny decision. Chapter 5 explores the identified synergy in antibiotic-resistant models, focusing on the impact of antibiotic resistance on the effect of combining temperate phages with antibiotics. While the majority of cases demonstrated synergy comparable to the absence of antibiotic resistance, an exception was noted in the acetylation-resistant models for both gentamicin and ciprofloxacin. These resistance genes abolished synergy with the temperate phage, emphasizing the importance of the resistance mechanism within temperate phage antibiotic synergy (tPAS). In conclusion, this thesis underscores the lack of interest in temperate phages for therapy and demonstrates a scalable strategy to overcome the major barriers to their use. I uncover the mechanisms underlying the synergy and show that these concepts are applicable even in the context of resistance to the synergizing antibiotic. These findings propose a remarkable shift in how antimicrobial therapy approaches are viewed. / Thesis / Doctor of Philosophy (PhD) / In the past decade, interest in viruses that only target bacteria (called “phages”) and their capacity to treat antibiotic-resistant infections has surged. Beginning with our systematic review on UTI treatments involving phages, we observed that none of the included studies explored the therapeutic use of the dormancy-capable “temperate” phages. This finding serves as motivation for the subsequent chapters of the thesis. In Chapter 3 I discovered temperate phage-antibiotic synergy (tPAS). An inventive strategy involving antibiotics activates temperate phages, demonstrating substantial synergy in eliminating bacterial infections and offering a potential breakthrough against antimicrobial resistance. In Chapter 4 I found that this synergy extends beyond antibiotics that result in a response to DNA damage within the bacteria (SOS response) to include protein synthesis inhibitors, providing an innovative approach to combat bacterial infections. Finally, in Chapter 5 I extended the study to antibiotic-resistant models. Across a wide array of mechanisms for antibiotic resistance, all but two supported the synergy we observed with temperate phage. This highlights the importance of the resistance mechanism in temperate phage antibiotic synergy (tPAS). The finding of this thesis paves the route for potentially integrating temperate phages into mainstream medical practices alongside antibiotic interventions.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

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>

Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (&phi;SPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (&phi;SPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.

Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (&phi;SPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (&phi;SPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.

Staphylococcus aureus is a common bacterium found on the skin and mucosal membranes of about 20% of the population. S. aureus growth on the skin is harmless, but if it bypasses the skin it can causes life-threatening diseases such as pneumonia, meningitis, bacteremia, and sepsis. Antibiotic-resistant strains of S. aureus, called Methicillin Resistant S. aureus (MRSA), are resistant to most antibiotics except vancomycin. However, vancomycin resistant strains of MRSA are becoming more common. In this study, 12 phages were isolated capable of infecting human S. aureus and/or MRSA strains. Five phages were discovered through mitomycin C induction of prophages and seven phages were found through enrichment of environmental samples. Primary S. aureus strains were also isolated from environmental sources to be used as tools for phage discovery and isolation as well as to examine the target cell host range of the phage isolates. S. aureus isolates were tested for susceptibility to oxacillin in order to determine methicillin-resistance. Experiments were performed to assess the host range and killing potential of newly discovered phage. The M1M4 phage had the broadest host range and lysed 12% of the S. aureus strains that were tested. The host ranges were reinforced by spectrophotometric assay data which showed a reduction in bacterial optical density of 1.3 OD600. The phages were used to decontaminate MRSA from fomites (glass and cloth) and successfully reduced colony forming units by 1-2 logs, including tests of a phage cocktail against a cocktail of MRSA isolates. Our findings suggest that phage treatment can be used as an effective tool to decontaminate human MRSA from both hard surfaces and fabrics.

The bacterial flagellum is a rotary motor that propels motile bacteria through their surroundings via swimming motility, or on surfaces via swarming motility. The flagellum is a key virulence factor for motile pathogenic bacteria. Viruses that infect bacteria via this appendage are known as flagellotropic or flagellum-dependent bacteriophages. Much like other phages, flagellotropic phages are of interest for clinical applications as antibacterial agents, particularly against multidrug resistant (MDR) bacteria. Bacteriophage χ is a flagellotropic phage that infects multiple species of motile pathogens. In the projects described below, we characterized several aspects of the complex interactions between χ and two of its hosts: Salmonella enterica and Serratia marcescens. In Chapter I, we describe in detail the existing knowledge on flagellum-dependent bacteriophages, pathogenic bacteria, and the flagellar motility system. We also expand significantly on flagellotropic phage χ. In Chapter II, we describe our discovery of S. enterica cellular components other than motility that are crucial for bacteriophage χ infection, making the key discovery that the AcrABZ-TolC multi-drug efflux system is required for infection to proceed. We additionally found that the host molecular chaperone trigger factor is important for the χ phage lifecycle. In Chapter III, we outline our characterization of the initial binding interaction between χ and the flagellum, determining that of flagellin's seven domains, C-terminal domain D2 is the most important for χ adsorption. In Chapter IV, we expand on this by discussing our work that determined that the χ tail fiber protein is encoded by the gene CHI_31, purification of this recombinantly-expressed protein, and demonstration of its direct interaction with the flagellar filament. Lastly, in Chapter V, our findings indicate that S. marcescens is able to detect χ infection and lysis in the surroundings and alter gene expression, resulting in an increase in the production of the red pigment prodigiosin. Overall, our hypothetical model for χ infection is as follows: χ binds to the flagellum of its host using its single tail fiber, composed of monomers of the CHI_31 gene product gp31. This tail fiber interacts with CTD2 of flagellin, and the rotation of the flagellum brings the phage to the cell surface, where it interacts with AcrABZ-TolC to inject its genetic material into the host cytoplasm. At some point during the process of production of phage particles and subsequent cell lysis, the host molecular chaperone trigger factor likely assists with proper folding of χ proteins. After cell lysis, cells in the surroundings are capable of detecting lysis and responding accordingly, at least in the case of S. marcescens. This research is clinically relevant for a number of reasons. Phage therapy, the use of bacteriophages as antibacterial agents, requires knowledge of phage infection pathways for optimal implementation. The fact that the flagellum and a complex mediating MDR are both essential for χ infection leads to particular interest in χ for this application. Knowledge of the host-determining factors between χ and Salmonella may lead to the ability to alter the χ phage genome to target specific pathogenic Salmonella or Escherichia coli strains while avoiding disruption of beneficial bacterial communities. / Doctor of Philosophy / Bacteriophages (phages) are viruses that only infect bacteria. They do not harm animal cells or the human body, despite being highly effective predators of bacteria. As such, they have applications in the medical field as antibacterial agents, similar to antibiotics. Phages that infect pathogenic bacteria like Salmonella are of particular interest for scientific research. Bacteriophage χ (Chi) infects bacteria by binding to their flagella, propeller-like appendages that a bacterial cell uses to swim through its surroundings. In many bacterial species, flagella and the ability to swim are closely involved in human infection. Due to this, flagellotropic (flagellum-dependent) phages like χ may be particularly useful as antibiotics. Throughout this project, we characterized the χ phage infection process, including exploring how it attaches to flagella, interactions it has on the surface of and inside Salmonella cells, and the largely unexplored relationship with Serratia marcescens, another bacterial species that causes illness in humans and is highly antibiotic resistant. Overall, our research contributes to the medical field, and indicates that χ may serve as a highly effective antibacterial treatment.

L'utilisation de virus de bactéries ou bactériophages pourrait être un complément efficace à l’antibiothérapie. Mon travail a porté sur la caractérisation de bactériophages dirigés contre l’espèce Pseudomonas aeruginosa, pathogène opportuniste responsable d'infections des voies respiratoires des patients atteints de mucoviscidose.J'ai tout d'abord déterminé la sensibilité des souches mucoviscidosiques au Pyophage (un cocktail de phages thérapeutiques Géorgien) et identifié six phages lytiques de quatre genres différents. Environ 15% des souches sont résistantes au Pyophage. Ensuite, en utilisant les souches cliniques multi-résistantes aux phages comme bactérie d’enrichissement, 32 phages ont été obtenus à partir des eaux usées de France et Côte d’Ivoire. Tous les phages analysés sont caudés et distribués au sein de dix genres parmi lesquels six exclusivement lytiques. J'ai identifié des souches bactériennes qui demeurent insensibles à tous les phages. J'ai montré que le système CRISPRs-Cas n'est pas associé à la résistance des souches aux phages lytiques. / The use of viruses of bacteria commonly called bacteriophages could constitute an efficient complement to antibiotics. During my PhD, I have characterized phages infecting the opportunistic pathogen Pseudomonas. aeruginosa, responsible for lung infections in cystic fribrosis patients. Firstly, I investigated the efficiency of Pyophage (a cocktail of phages therapeutic Georgian) on clinical P. aeruginosa strains and recovered six lytic phages from four different genus. The Pyophage appears to be unactive on approximately 15% of clinical strains. Secondly, and using multi-phages resistant strains as enrichment bacteria, 32 phages were isolated from waste water of France and Côte d’Ivoire. All phages are tailed and distributed within ten different genus including six exclusively lytic. I identified bacterial strains which remain insensitive to all phages. I also demonstrated that the CRISPRs-cas system plays no role in the resistance of strains to lytic phages.