Isolation and Characterization of the Polyvalent Enterobacteria-Infecting Phage Cit2 with Potential for Biocontrol Applications

<i>Salmonella enterica</i> is a major foodborne pathogen globally, often associated with poultry and fresh produce. The rising prevalence of multidrug-resistant (MDR) strains and the limited efficacy of conventional decontamination methods highlight the need for alternative, targeted strategies. Bacteriophages (phages) have emerged as promising biocontrol agents due to their specificity, safety, and potential for direct application in food systems. In this study, we isolated and characterized the polyvalent lytic phage <i>Citrobacter</i> phage vB_CfrD-Cit2 (Cit2) and evaluated its effectiveness against <i>S. enterica</i> in two food matrices: chicken meat and lettuce. Cit2 belongs to the <i>Tequintavirus</i> genus and showed lytic activity against <i>Citrobacter freundii</i>, <i>Shigella flexneri</i>, and different <i>S. enterica</i> serovars. Genomic analysis confirmed the absence of virulence, antibiotic resistance, or lysogeny-related genes, supporting its classification as genetically safe. The phage remained stable across a broad range of pH values and temperatures. In biocontrol assays, Cit2 significantly reduced <i>S. enterica</i> serovar Enteritidis counts on lettuce at room temperature by 1.83, 1.84, and 1.55 log₁₀ CFU/mL after 1, 2, and 24 h, respectively. In refrigerated chicken meat (4 °C), reductions of 0.79, 0.84, 0.76, and 0.69 log₁₀ CFU/mL were observed at 1, 6, 24, and 48 h post-treatment, respectively. Phylogenetic and protein identity analyses suggest that Cit2 likely targets the outer membrane receptor FhuA, similarly to phage T5. Future studies should aim to optimize multiplicity of infection (MOI) and cocktail formulations to improve host range and efficacy, as well as assess phage stability across different food matrices. Taken together, these findings support Cit2 as a promising candidate for the development of phage-based interventions to enhance food safety.