Beyond Antibiotics: Building a Pseudomonas aeruginosa Phage Library for Targeted Therapy

Abstract

Background: Microbial resistance due to antibiotic misuse resulted increasing illness and mortality rates. Pseudomonas aeruginosa, a common hospital-acquired bacterium, has become highly resistant globally, including Lebanon where carbapenem-resistant strains are now prevalent. This resistance has led to higher mortality rates, longer hospital stays, and increased healthcare costs. Consequently, there is a pressing need for alternative solutions, and phage therapy, which consists of using viruses that specifically target and lyse bacterial cells, is being reconsidered as a potential alternative approach. In this study, we aim to isolate and characterize lytic phages from natural sources to evaluate their efficacy against multidrug-resistant Pseudomonas aeruginosa strains, to develop a phage library for future therapeutic interventions.

Methods: Screening for phages will be conducted by mixing natural resources samples (in our case 12 sewage samples from different regions across Lebanon) and the bacteria in a 96 well plate and OD will be measured. Then, a Double Agar Layer assay will be performed to isolate the phages. Once isolated, phages will be spotted over different Pseudomonas aeruginosa strains for a host range determination. Additionally, phages will be characterized by their bacteriolytic activity, their latency period and burst size determined by a one-step growth assay, their ability clear bacterial biofilm post formation and their stability under different temperature and pH values. Finally, a DNA extraction and sequencing will be performed to identify the phage whole genome.

Results: Six distinct phages were isolated against three Pseudomonas aeruginosa strains from Al Nabatieh and Iaat sewage samples with a titer ranging between 1015 and 1040 PFU/ml. Three phages recorded the widest host range and showed considerable bacterial killing effect, while the rest were more host specific. Only two phages out of these three presented a potential role in significantly eliminating mature biofilm. PSA 44 NAB 02 demonstrated excellent pH and thermal stability and it adsorbed to its host within 5 minutes and lysed the cell within 30 minutes, releasing approximately 30 virions per bacterial cell.

Conclusion: We successfully isolated and characterized six bacteriophages. Several phages, highlighted by PSA 44 NAB 02, are potential candidates to be included in our phage library giving there promising bacterial killing capacity and ability in clearing mature biofilm.