A Programmable CRISPR-Phage Strategy to Disable Antibiotic Resistance and Biofilm Formation in Pseudomonas aeruginosa

Antibiotic resistance has become an impending global crisis in modern medicine, and Pseudomonas aeruginosa is recognized by the World Health Organization (WHO) as a critical priority pathogen. In its published list of antibiotic-resistant bacteria requiring urgent development of new antibiotics, P. aeruginosa ranks among the highest due to its prevalence, the difficulty of treating it in clinical settings, and its prominent role in nosocomial infections, notably ventilator-associated pneumonia, bloodstream infections, and chronic lung disease in cystic fibrosis patients. This bacterium uses overlapping layers of defense that include β-lactamase production, efflux pumps, changes in porins, and quorum-sensing systems such as lasI/lasR that regulate biofilm formation, which can make the bacteria up to a thousand times more tolerant of antibiotics than planktonic cells. Current therapeutic options from β-lactam/inhibitor combinations and phage cocktails to quorum-sensing inhibitors are gradually losing efficacy, as each addresses only one aspect of resistance without fully eliminating resistance determinants or preventing biofilm maturation. Engineered bacteriophages with dual functions, bacterial lysis and CRISPR-based disruption of targets such as ampC and lasI, could therefore provide a comprehensive approach to counter both antibiotic resistance and biofilm development, paving the way for more effective treatments against multidrug-resistant Pseudomonas aeruginosa. Accordingly, the objective of this study is to evaluate whether bacteriophage-mediated delivery of a dual CRISPR–Cas9 system targeting ampC and lasI can simultaneously enhance β-lactam antibiotic susceptibility and reduce biofilm formation in Pseudomonas aeruginosa.