(1) Background: The emergence of multidrug-resistant (MDR) Salmonella enterica poses a major threat to global public health, underscoring the urgent need for alternative therapeutic strategies. Bacteriophages represent a promising alternative due to their high specificity and potent ability to lyse MDR strains. (2) Methods: In this study, we isolated a novel MDR Salmonella Enteritidis-targeting bacteriophage from Lebanese sewage and characterized its host range, thermal and pH stability, and infection dynamics. Whole-genome sequencing was performed using Illumina technology to determine its genetic features and taxonomic classification. (3) Results: the bacteriophage was classified within the genus Jerseyvirus and the class Caudoviricetes with a 43 kb dsDNA genome encoding 66 open reading frames (ORFs). It demonstrated remarkable thermal stability, retaining infectivity after prolonged incubation at 65 °C, and showed a broad host range. The phage formed large, clear plaques, displayed rapid adsorption (>97% within 3 min), a short latent period (20 min), and a burst size of ~32 PFU per cell. Genome analysis revealed no lysogeny, virulence, or resistance genes, confirming its strictly lytic nature and supporting its potential use as a biocontrol agent. (4) Conclusions: These findings identify SA01 as a novel, strictly lytic, and thermally stable bacteriophage with strong potential as a biocontrol agent against multidrug-resistant Salmonella Enteritidis. Its broad host range suggests potential activity also against other Salmonella enterica serovars, supporting its applicability in food safety and biotechnology.

1) Background: The emergence of multidrug-resistant (MDR)-Salmonella enterica poses a major threat to global public health underscoring the urgent need for alternative therapeutic strategies where bacteriophages are a promising candidate due to their high specificity and potent ability to lyse MDR strains.2) Methods: In this study, we isolated a novel MDR-Salmonella Enteritidis-targeting bacteriophage from Lebanese sewage and characterized its host range, thermal and PH stability, and infection dynamics. Whole-genome sequencing was performed by Illumina to determine genetic features and taxonomic position.3) Results: the bacteriophage belongs to genus Jerseyvirus and Class Caudoviricetes with a 43kb dsDNA genome and 66 ORFs. It demonstrated extraordinary thermal stability, retaining infectivity after prolonged incubation at 65 °C, showed a broad host range, produced large, clear plaques, displayed rapid adsorption (>97% within 3 min), a short latent period (20 min), and a burst size of ~320 PFU per cell. Genome analysis revealed no lysogeny, virulence, or resistance genes, confirming its strictly lytic nature and supporting its potential use as a biocontrol agent. 4) Conclusions: These findings identify SA01 as a novel, strictly lytic, and thermally stable bacteriophage with strong potential as a biocontrol agent against multidrug-resistant Salmonella Enteritidis, supporting its potential activity also against other Salmonella enterica serovars for future applications in food safety and biotechnology.

Salmonella enterica serovar Enteritidis is a leading cause of foodborne zoonoses worldwide. The rapid emergence of multidrug-resistant (MDR) strains has compromised traditional antimicrobial therapies, necessitating the development of biosafe alternatives such as bacteriophages. This study aimed to isolate and comprehensively characterize novel lytic bacteriophages targeting multidrug-resistant Salmonella enterica subspecies enterica serovar Enteritidis isolates from Lebanon. In this study, four novel Salmonella phages, EDA02, EDA03, EDA05, and EDA06, were isolated from wastewater and poultry effluents in Lebanon. The isolates were characterized using host range profiling, one-step growth kinetics, and physicochemical stability assays. Comprehensive whole-genome sequencing (WGS) and phylogenetic analyses were performed to assess their genomic safety and taxonomic placement. Phages EDA03 and EDA06 exhibited the broadest intra-serovar lytic activity within the tested panel, infecting up to 72% and 67% of the MDR isolates, respectively. One-step growth analysis revealed latent periods of 30–40 min, with burst sizes ranging from 6.0 to 150 phages/infected cell. All four phages demonstrated robust stability across pH 4.7–10.3 and temperatures from 4 °C to 50 °C. WGS revealed genome sizes ranging from 42.3 kb to 108.8 kb, with no identified genes associated with lysogeny, virulence, or antimicrobial resistance. Phylogenomic analysis assigned all isolates to the family Straboviridae, with <95% intergenomic similarity to their closest RefSeq relatives, supporting their classification as novel species. The isolated phages demonstrate substantial lytic activity and environmental resilience under the tested conditions. Their complementary lytic profiles, environmental resilience, and genomic safety support their further evaluation as biocontrol candidates. This study represents the first genomic and phenotypic characterization of anti-Salmonella Enteritidis phages from Lebanon. These findings support the development of phage-based interventions for food safety and antimicrobial resistance mitigation in resource-limited settings.