Antibacterial Activity of a Fused Endolysin <scp>ENDO</scp> ‐1252/ <scp>KL9P</scp> Against Multiple Serovars of <i>Salmonella enterica</i>

Salmonella enterica (SE) is one of the most prevalent enteric pathogens globally and infects humans through contaminated food and water sources. The rising trend of antibiotic-resistant SE strains poses a critical threat to public health. Bacteriophage-encoded endolysins evolve a promising alternative as antimicrobial agents for combating SE infections. These enzymes target the peptidoglycan layer of bacterial cells, causing cell lysis and death. However, the use of endolysins against Gram-negative bacteria is challenging due to the composition of the outer membrane, which acts as a barrier preventing the endolysins from reaching the peptidoglycan layer. KL9P is a short amphipathic peptide containing both hydrophobic and hydrophilic regions, enabling it to interact with membranes and aqueous environments. In this study, an endolysin ENDO-1252, a Salmonella bacteriophage-encoded enzyme, was fused with a short peptide KL9P and produced an advanced endolysin, ENDO-1252/KL9P, which enhanced its ability to lyse multiple serovars of SE. ENDO-1252/KL9P exhibited potent lytic activity against SE strains with optimal bactericidal effects observed at 20 μM and incubation at 37°C in 20 mM HEPES buffer (pH 7.4). The lytic activity of this endolysin was also evaluated under various conditions, including pH ranges and temperatures, revealing that ENDO-1252/KL9P retained significant lytic activity across a range of temperatures (25°C-40°C) and pH values (6.0-9.0). The fusion protein demonstrated the highest lytic efficiency against SE serovars, specifically S. Enteritidis, S. Heidelberg, and S. Pullorum. Immunofluorescence analysis confirmed the binding of ENDO-1252/KL9P to the bacterial cell wall, indicating the co-localization with the peptidoglycan layer. These results suggest that ENDO-1252/KL9P is a promising antibacterial agent inhibiting predominant serovars of SE, showing enhanced lytic activity without outer membrane permeabilizers.