CRISPR-Engineered Phages Displaying LL37 Peptide Show Enhanced Salmonella Killing

The global rise of antibiotic-resistant bacteria, particularly Salmonella, poses a severe threat to public health, leading to treatment failures and increased mortality. Traditional antibiotics are becoming less effective, necessitating novel therapeutic strategies. Phage therapy, utilizing bacteriophages (viruses that specifically infect and kill bacteria), offers a promising alternative. However, phages can be further optimized. This study addresses the knowledge gap of whether genetically engineering phages to display antimicrobial peptides can significantly boost their antibacterial efficacy against resistant strains.

The CRISPR/Cas9-engineered phage demonstrated significantly enhanced antibacterial activity against Salmonella enterica serovar Typhimurium compared to both the unengineered phage and free LL37 peptide. The engineered phage achieved a remarkable 99.9% (3-log reduction) in Salmonella bacterial count within just 4 hours of exposure, which was a 2.5-fold greater reduction than observed with the unengineered phage (p<0.001). This enhanced bactericidal effect was also evident in biofilm disruption, where the engineered phage led to a 43% reduction in established Salmonella biofilms compared to the control group (p<0.01). Furthermore, analysis showed a 1.8-fold increase in bacterial membrane permeabilization markers, indicating a dual mechanism of action from both phage lysis and LL37-mediated membrane disruption.