STUDY OF A NOVEL SALMONELLA BACTERIOPHAGE AND ITS MECHANISM OF LYTIC EFFECT AGAINST MULTIPLE SEROVARS

Abstract

Salmonella enterica (S. enterica) is a leading cause of foodborne illness worldwide, resulting in substantial economic and public health challenges. The emergence of antibiotic-resistant strains has heightened the need for innovative antimicrobial approaches. Bacteriophage (phage) therapies and phage-encoded enzymes such as endolysins offer promising alternatives to conventional antibiotics and address antimicrobial resistance issues. However, the limitation on the lysis ability of most phages is their species-specificity to the bacterium. S. enterica has various serovars, such as S. enterica Enteritidis and S. enterica Typhimurium, which are involved in gastrointestinal diseases globally and the United States. In this study, we focus on using a promising alternative, phage and phage-encoded endolysin, to inhibit the growth of multiple Salmonella serovars. A novel phage, Salmonella phage-1252 (Genbank ID: PP695294.1), was isolated and characterized. Whole-genome sequencing analysis revealed phage-1252 is a novel phage strain that belongs to the genus Duplodnaviria in the Myoviridae family and consists of a 244,421 bp dsDNA, with a G + C content of 48.51%. Its plaque diameters are approximately 2.5 mm to 0.5 mm on the agar plate. It inhibited Salmonella Enteritidis growth after 6 h. The growth curve showed that the latent and rise periods were approximately 40 min and 30 min, respectively. The burst size was estimated to be 56 PFU/cell. The phage remained stable and maintained its activity between 4 °C and 55 °C for up to 1 hour. To address phage limitations, we used endolysins, enzymes encoded by phages that target the peptidoglycan layer of bacterial cells, leading to their rupture and destruction. However, the application of phage-encoded endolysin against Gram-negative bacteria is limited due to the outer membrane in the cell wall, which hinders the permeation of externally applied endolysins. We successfully expressed and purified phage-encoded endolysin, ENDO-1252. It has intense lytic activity against S. Enteritidis and S. Typhimurium. In addition, ENDO-1252 showed optimal thermostability and lytic activity at 25 °C with a pH of 7.0. In combination with 0.1 mM EDTA, the effect of 120 µg of ENDO-1252 for 6 hours exhibited the highest lytic activity, reducing 1.15 log or 92.87% on S. Enteritidis. To enhance the antibacterial efficacy of ENDO-1252 without synergy with EDTA, the endolysin ENDO-1252 was engineered into a fusion protein, ENDO-1252/KL9P, by adding a short peptide, KL9P. This fusion protein exhibited potent lytic activity against multiple S. enterica serovars, with optimal activity at 37 °C and pH 7.4. ENDO-1252/KL9P maintained significant lytic activity across a broad pH range (6.0–9.0) and temperature (15 °C–45 °C). Immunofluorescence analysis confirmed its binding to bacterial peptidoglycan, indicating a direct interaction with the bacterial cell wall. The findings of this dissertation suggest that phage-1252 and its engineered endolysins could effectively reduce S. enterica in food production systems and help address antibiotic resistance challenges. In the future, we recommend exploring novel phages to evaluate their lytic activity against Salmonella, identifying peptides that enhance endolysin activity, and conducting in vivo studies to assess the safety and efficacy of phages and their derivatives for therapeutic applications. Homologous recombination could be employed to engineer mutant E. coli Nissle strains as probiotics capable of inhibiting Salmonella growth in human or animal guts.