Broad-spectrum lytic potential of endolysins derived from prophages of clinical Salmonella isolates

In the post-antibiotic era, alternative therapeutic strategies were urgently needed. Here, we characterized 514 prophages from 105 clinical Salmonella isolates, assessing their carriage of antimicrobial resistance (AMR) genes and virulence factors (VFs) to evaluate biosafety and evolutionary dynamics. Our analyses revealed significant correlations between the abundance of each prophage and host serotypes, as well as between prophage-borne AMR genes and host age or serotype background. Phylogenetic analysis showed that most prophages are related to known Salmonella phages, though a subset shares homology with Burkholderia viruses, suggesting inter-generic evolutionary connections. A comprehensive screening of prophage-encoded proteins identified a large repertoire of endolysins, with 80.93% of prophages carrying at least one such enzyme. Sequence-based clustering grouped these endolysins into seven families, three of which are widely distributed across the isolate collection. Structural modeling indicated that representative enzymes from these major groups are structurally analogous to thermostable, broad-spectrum lysozymes, which revealed the potential of the proteins as alternatives to antibiotics for treatment. To validate the therapeutic potential of prophage-derived lytic enzymes, we expressed the candidate endolysin Lys2823 and demonstrated its lytic activity against outer-membrane-permeabilized Salmonella. These results indicate that Lys2823 holds promise as a biocontrol agent for the prevention and treatment of Salmonella infections, thereby contributing to food safety and public health. This study provides experimental evidence supporting the development of prophage-derived endolysins as novel antimicrobial agents.