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.

Early function of a temperature-sensitive mutant of staphylophage 44A HJD was examined during the twenty-five-minute period following infection. Host cell and phage DNA were labeled with C and3H respectively. DNA was separated into linear and covalently closed circular (CCC) forms by density-gradient centrifugation. The host, S. aureus, shows no CCC DNA, and apparently carries no plasmid. Following infection with wild type phage, CCC DNA forms occur in tritiated and 1 C DNA fractions 10 to 15 min after infection. Infection with mutant at permissive temperature also demonstrates CCC DNA with both labels. Infection with mutant at nonpermissive temperature produced no CCC DNA during the first 25 min after infection. The impaired function in this mutant may be a linker protein.