Phage Journeys: Unraveling Prophage and Anti-phage systems Mobilisation in P. aeruginosa for Enhanced Phage Therapy Efficacy

Antimicrobial resistance (AMR) represents a significant global health challenge, threatening the effectiveness of existing antibiotics and complicating the treatment of infectious diseases. The increasing rate of emergence of multidrug-resistant (MDR) pathogens has created a pressing need for innovative approaches to combat AMR. Pseudomonas aeruginosa stands out as a prominent AMR pathogen, posing significant challenges in clinical settings. Known for its adaptability and intrinsic resistance mechanisms, this opportunistic pathogen thrives in diverse environments, including hospitals. MDR P. aeruginosa represents one of the main causes of ventilator-associated pneumonia (VAP), a condition that affects 5-40% of intensive care units (ICU) patients that undergo mechanical ventilation for more than 48 hrs. P. aeruginosa ability to form biofilms in endotracheal tubes that are difficult to eradicate favours its seeding into the patients’ airways and lungs. The remarkable resistance that P. aeruginosa exhibits to diverse antibiotic classes renders its eradication in VAP patients challenging and, in many cases, leads to recurring infections, longer hospitalisation periods, increased economic toll on health services and high mortality rates. Phage therapy is emerging as a promising solution to combat AMR infections. In several trials phage cocktails have demonstrated efficacy in targeting MDR P. aeruginosa strains and eradicating biofilms in VAP patients, thus rendering phage-based therapies a promising alternative route to treat VAP patients. However, the evolutionary arms race between bacteria and phages has yielded a diverse and broad arsenal of phage defence systems within bacterial populations. Recent research has identified approximately 200 unique anti-phage systems, highlighting the intricate dynamics between bacteria and phages during infection. P. aeruginosa harbours an extensive repertoire of anti-phage systems, with certain strains carrying up to 17, conferring resistance against most known Pseudomonas-targeting phages. Additionally, anti-phage systems are often carried on mobile genetic elements (MGE), such as prophages. These represent genomic locations that can be frequently exchanged across bacterial strains of the same species through horizontal gene transfer. Localisation of defence systems on MGEs favours their mobility in mixed populations of Pseudomonas strains at the site of infection, provide population-wide protection against a wide diversity of phages. The prevalence of these defence mechanisms in Pseudomonas and their mobilisation across strains poses a significant obstacle for phage therapy, potentially compromising the efficacy of phage cocktails against heterogeneous populations of P. aeruginosa strains commonly encountered in VAP infections. It is therefore of paramount importance to understand how defence systems, their interaction network, and their mobility across heterogeneous populations can affect the efficacy of phage therapy and its promise in combating AMR infections in VAP patients. With this project, I will address this knowledge gap by utilising an endotracheal tube biofilm model to investigate P. aeruginosa MGE-encoded anti-phage systems' mobility and competition against phage cocktails in a physiologically relevant context. The data and insights obtained in this study will be leveraged to explore routes for engineering and designing phage-based therapies with enhanced efficacy. These therapies will be tailored to each patient and capable of circumventing anti-phage systems, providing an effective and potent tool to combat AMR. Furthermore, this study will provide a high-throughput platform that can be extended to investigate other VAP-relevant pathogens, poly-microbial infections, and the impact of the synergistic interactions of multiple anti-phage systems encoded in specific pathogens on the efficacy of phage cocktails.