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

## TL;DR

A new bacteriophage called Cit2 was isolated and shown to effectively reduce Salmonella in food matrices like chicken meat and lettuce, offering a potential biocontrol solution.

## Contribution

The isolation and characterization of Cit2, a polyvalent lytic phage with broad activity against Salmonella and other pathogens, is presented.

## Key findings

- Cit2 significantly reduced Salmonella Enteritidis on lettuce by up to 1.84 log10 CFU/mL within 2 hours.
- The phage Cit2 remained stable across various pH and temperature conditions, indicating robustness for food applications.
- Phylogenetic analysis suggests Cit2 targets the outer membrane receptor FhuA, similar to phage T5.

## Abstract

Salmonella
enterica 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 Citrobacter phage vB_CfrD-Cit2 (Cit2) and evaluated its
effectiveness against S. enterica in
two food matrices: chicken meat and lettuce. Cit2 belongs to the Tequintavirus genus and showed lytic activity against Citrobacter freundii, Shigella flexneri, and different S. enterica 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 S. enterica 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.