Improved Pseudomonas aeruginosa detection through the development of an engineered reporter bacteriophage

Currently, there is a critical need for the rapid and accurate detection of Pseudomonas aeruginosa, a major pathogen responsible for nosocomial infections and high mortality rates due to its antibiotic resistance and virulence. To address this challenge, a new method is here described based on the development of a genetically engineered reporter bacteriophage that expresses the NLuc luciferase upon bacterial infection. The NLuc luciferase gene was inserted in the previously characterized vB_PaeP_PE3 P. aeruginosa phage, using the yeast-based phage-engineering platform. The expression of the NLuc luciferase, driven by endogenous phage promoters, ensures high-level reporter expression during bacterial infection, producing a strong luminescence signal upon substrate addition. The ability to detect viable P. aeruginosa cells as few as 10^2 CFU/mL within 7 h and approximately 1 CFU/mL within 24 h with a 100 % specificity, significantly reduces the turnaround time compared to traditional methods. Moreover, the method was successfully validated in artificially contaminated blood samples, highlighting its potential for clinical applications in diverse settings, including resource-limited areas due to its simplicity and reduced costs. This innovative approach promises to improve P. aeruginosa diagnosis, facilitating timely and appropriate antimicrobial therapy.