Characterization of a lytic Salmonella phage GF04 within the Jerseyvirus lineage and identification of yjiK_2 as a candidate phage receptor.

INTRODUCTION: Salmonella enterica is a major antimicrobial-resistant pathogen recognized by the World Health Organization, with high infection and mortality rates largely driven by antibiotic overuse. Bacteriophage therapy has emerged as a promising alternative due to phages' high specificity, self-replication at infection sites, and safety when properly selected; however, its success depends on understanding phage-host interactions, particularly receptor recognition and resistance mechanisms.

METHODS: Salmonella phage GF04 was isolated from sewage using S. enterica serovar Enteritidis strain SAL271 as host and purified through successive plaque isolation. Phage characterization included adsorption kinetics, one-step growth curve analysis, bacteriolytic activity across multiple MOIs, and host range determination using efficiency of plating (EOP) on 19 clinical isolates. Whole genome sequencing and annotation were performed to assess taxonomic placement and the presence of virulence, antimicrobial resistance, and lysogeny-associated genes. A spontaneous phage-resistant derivative was isolated following phage exposure and analyzed by comparative whole genome sequencing and adsorption assays. Structural effects of identified mutations were evaluated using AlphaFold modeling.

RESULTS: GF04 forms clear plaques and exhibits rapid adsorption, with most virions attaching within 5 minutes. One-step growth analysis revealed a ~45-min latent period and a burst size of ~103 PFU per infected cell, indicating efficient replication. GF04 suppressed bacterial growth across multiple multiplicities of infection and demonstrated a relatively broad host range within the tested isolates, with clear strain-dependent variability, infecting 14 of 19 S. Enteritidis isolates as determined by efficiency of plating (EOP) analysis. EOP values ranged from no detectable infection to high efficiency (up to 0.8 relative to the reference host). Whole genome sequence analysis confirmed the absence of detectable lysogeny, virulence, or antimicrobial resistance genes, and phylogenetic analyses demonstrated that GF04 is a new species within the Jerseyvirus lineage (family Sarkviridae). Furthermore, a spontaneous phage-resistant variant of SAL271 exhibited a complete loss of phage adsorption compared to the wild-type strain. Comparative genomic analysis subsequently identified a fixed missense mutation (P128L) in the predicted membrane-associated gene yjiK_2. Structural modeling suggested that this mutation induces localized conformational changes potentially affecting receptor accessibility.

DISCUSSION: Comparative analysis of the resistant derivative implicates the mutation in yjiK_2 as the most likely genetic basis of resistance, potentially underlying the observed loss of phage adsorption. This suggests that yjiK_2 may encode or contribute to a receptor-associated determinant involved in GF04 adsorption. Together, these findings support the lytic potential and preliminary therapeutic relevance of GF04 while identifying yjiK_2 as a candidate phage-resistance determinant that warrants further functional validation.