# Genomic and Functional Characterization of Lytic Tlsvirus Bacteriophages Targeting Salmonella Infantis Isolated from Poultry Farms in Ecuador

## TL;DR

This study identifies three bacteriophages that can safely and effectively target antibiotic-resistant Salmonella Infantis in poultry, offering a potential alternative to antibiotics.

## Contribution

The discovery and characterization of three lytic bacteriophages with high efficacy and safety for biocontrol of Salmonella Infantis in poultry.

## Key findings

- Three bacteriophages (GS71, GS156, GS166) were isolated that rapidly kill Salmonella Infantis and are stable under harsh conditions.
- Two of the phages (GS71 and GS166) effectively lyse most S. Infantis strains, while none of the phages carry harmful genes.
- The phages are genetically similar and belong to the Tlsvirus genus, with conserved genes for replication and lysis.

## Abstract

Salmonella causes millions of foodborne illnesses each year, and the rise of strains that no longer respond to antibiotics makes it necessary to find new ways to control this bacterium in the food chain. In Ecuador, the type of Salmonella most commonly found in poultry farms, poultry meat, and human infections is Salmonella Infantis. In this study, we searched for viruses that specifically infect and destroy this bacterium, known as bacteriophages, to evaluate whether they could help improve food safety. We isolated three bacteriophages from chicken farms in different regions of Ecuador and found that they acted quickly, produced many new viral particles, and tolerated high temperatures and alkaline conditions. Although the bacteriophages came from different places and times, they were genetically very similar, reflecting the low diversity of Salmonella Infantis in Ecuador. Two of them were able to kill most of the tested bacterial strains. None carried genes associated with harmful effects. These results show that the three bacteriophages are safe and promising candidates for use in mixtures designed to reduce antibiotic-resistant Salmonella Infantis in poultry production.

Salmonella is responsible for millions of foodborne illnesses worldwide. The emergence of antibiotic-resistant Salmonella strains necessitates the development of alternatives for controlling this microorganism in the food supply chain. In Ecuador, Salmonella Infantis (S. Infantis) is the most frequently isolated serovar in poultry farms, poultry food products, and human infections. The objective of this study was to isolate and characterize lytic bacteriophages against a S. Infantis strain from poultry products in Ecuador to evaluate their potential for biocontrol. Three bacteriophages, GS71, GS156, and GS166, were isolated from chicken feces samples and showed short latent times (5–10 min), burst sizes of 205–231 PFU/cell, and stability up to 50 °C and pH = 10. Despite being isolated at different times and locations, they exhibited high genomic similarity (91.9–98.7%), reflecting the low diversity of Ecuadorian S. Infantis strains. VIRIDIC and phylogenetic analyses placed them within the Tlsvirus genus, showing conserved gene modules for replication, morphogenesis, and lysis. Putative endolysin and depolymerase genes were identified, supporting their anti-biofilm activity against biofilm-forming bacteria. Host range assays showed GS71 and GS166 lysed most S. Infantis field strains, whereas GS156 had a narrower spectrum linked to a unique polynucleotide kinase insertion. TEM confirmed Siphovirus-like morphology with icosahedral capsids (~55 nm) and long non-contractile tails. No genes associated with lysogeny, virulence, or antibiotic resistance were found. These findings support GS71, GS156, and GS166 as safe and effective candidates for bacteriophage cocktails targeting multidrug-resistant S. Infantis in poultry production.