Genomic and Functional Characterization of lytic Tlsvirus bacteriophages targeting Salmonella Infantis isolated from poultry farms in Ecuador

Genomic and Functional Characterization of lytic Tlsvirus bacteriophages targeting Salmonella Infantis isolated from poultry farms in Ecuador | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genomic and Functional Characterization of lytic Tlsvirus bacteriophages targeting Salmonella Infantis isolated from poultry farms in Ecuador Sandra Sevilla-Navarro, Ignacio Gómez-Cano, Ivette Castillo-Beckmann, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7411219/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacteria of the Salmonella genus are 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 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 Salmonella 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 tp 50ºC 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 strong anti-biofilm activity observed in vitro . 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 phage cocktails targeting multidrug-resistant S. Infantis in poultry production. Salmonella bacteriophage depolymerase biofilm biocontrol endolysin Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION Salmonella -related illnesses are a major global problem concern. Non-typhoidal Salmonella is estimated to cause 99.8 million illnesses and 155,000 deaths each year worldwide (Majowicz et al. 2010 ). Most human infections result from the consumption of undercooked contaminated food, especially poultry (Antunes et al. 2016 ). Studies have shown that, in Ecuador, Salmonella enterica serovar Infantis ( S . Infantis) is the most prevalent serovar in poultry farms, food, and humans, and it also exhibits multiple patterns of antibiotic resistance (Mejía et al. 2020). A similar trend is observed in other regions, such as Europe, where S. Infantis is among the top five European Union-acquired serovars involved in human infections. Moreover, it is by far the dominant serovar in the "broilers–broiler meat" category and ranks among the top four serovars across all considered food-animal sources (EFSA 2024). Bacteriophages (phages) are viruses that infect bacteria and possess several characteristics, such as high host specificity, the ability to replicate rapidly within bacterial cells, and the capacity to lyse their host upon completing their replication cycle. They are widely distributed in nature, particularly in environments rich in bacteria such as soil, water, and the human gut. Due to their specificity and bactericidal activity, phages have gained attention as potential alternatives to antibiotics, especially in the context of increasing antimicrobial resistance. Interest in phages as an alternative to antibiotics on farms has skyrocketed since the FDA approved the first bacteriophage product (phage) for food processing plants in 2006, and the EU approved anti- Salmonella phages for use in meat products in 2011. The criteria for selecting phages for application in agricultural facilities is that they do not have a lysogenic cycle, carry antibiotic resistance genes, or have pathogenic genes. Considering that 99% of bacteria persist in nature in biofilms(Yan & Bassler 2019 ) a desirable characteristic is that the phages carry depolymerases capable of attacking the biofilms that shelter and protect the bacteria. In this way, the phages, in addition to attacking bacteria, will eliminate them from the biofilms that act as reservoirs. In recent decades, there has been a notable increase in the prevalence of multidrug-resistant Salmonella worldwide, with S. Infantis emerging as one of the most concerning serovars (Mattock et al. 2024 ). In this context, phage therapy represents an alternative to antibiotics for controlling Salmonella proliferation and preventing human infections (Khan & Rahman 2022 ). Further, their ability to adapt alongside bacterial evolution makes them especially valuable in dynamic farm environments (Cui et al. 2024 ). Limited number of studies have been published regarding the use of bacteriophages in controlling Salmonella Infantis, yet their potential as biocontrol agents is promising (Battistelli et al. 2024 ; Sevilla-Navarro et al. 2024 b; Sevilla-Navarro et al. 2024 c). However, due to the genetic plasticity and high dissemination potential of this bacterium, as well as the presence of different S. Infantis clades circulating globally, it is essential to have previously isolated and well-characterized bacteriophages that are effective against region-specific strains. This would enhance the efficacy of phage-based interventions and support their targeted application in different epidemiological contexts. Beyond basic phenotypic characterization, understanding the genomic relationships between candidate phages is critical. Comparative genomics, phylogenetic analyses, and synteny mapping provide insights into their evolutionary stability, functional modules, and potential host-range determinants (Sattar 2022). Moreover, because bacterial resistance to phages can emerge through coevolutionary mechanisms, it is important to anticipate such dynamics when evaluating long-term efficacy (Oechslin 2018 ). In this context, the purpose of this study was to isolate and comprehensively characterize lytic phages targeting a Salmonella Infantis strain isolated in Ecuador, evaluating their biological properties, genomic safety, anti-biofilm potential, and phylogenetic relationships, for potential use as biocontrol agents in food supply chain MATERIALS AND METHODS Bacterial Strains and Culture Conditions S. Infantis U1068 was isolated in Quito, Ecuador, from chicken carcasses and used as a host for bacteriophage isolation. Additionally, strains of S. Infantis ATCC 51741, Salmonella enterica serovar Enteritidis ( S . Enteritidis) ATCC 13076, and Escherichia coli DH5α were utilized to visualize the morphology of the plaques of phages. Bacteria were incubated until exponential phase (OD 600 = 0.5–0.6); 100 µl of Salmonella was added to 100 µl of lysate at 10 3 pfu/ml. The mixture was incubated at 37°C for 5 minutes and then cultured using the double agar technique. The plaques were incubated at 37°C overnight. All strains were grown in liquid Trypto-Casein Soy Broth (TSB) medium (Difco™, BD, USA) at 37°C and 150 rpm. Cultures on solid medium were performed on TSB plates with 1% agar (Difco™, BD, USA). The S. Infantis strain used to propagate the phages was grown in liquid TSB medium with 0.2% MgSO 4 (Sigma-Aldrich, USA) at 37°C and 150 rpm. Isolation and Propagation of Bacteriophages Salmonella bacteriophages were isolated according to a previously described method (Kosznik-Kwaśnicka et al., 2020) with some modifications. Forty-eight chicken feces and wastewater samples were collected from rural areas of the Ecuadorian coast. The samples were enriched by diluting 3 g or 3 ml of the sample in TSB culture medium containing 0.2% MgSO 4 in a 1/10 ratio. Then, 1 ml of exponential-phase S . Infantis U1068 (OD 600 = 0.5–0.6) and ceftriaxone (CRO) at a final concentration of 10 ppm were added. The enriched samples were incubated at 37°C and 150 rpm overnight. The samples were centrifuged at 4000 rpm for 30 min at 4°C to separate bacteria and solid residues. The supernatant was filtered through a 0.22 µm syringe filter (Merck Millipore, USA), and chloroform was added at a ratio of 1/100. Ten µl of sample was mixed with 100 µl of S . Infantis, and the solution was cultured by the double-layer agar (DLA) method, using TSB with 0.2% MgSO 4 and 0.5% agar as the upper semisolid medium. The plates were incubated at 37°C and then examined for the presence of lysis plaques (circular clearing spaces) in the bacterial lawn. An isolated plaque from each sample was pricked with a pipette tip and suspended in 300 µl of TSB, 0.2% MgSO 4 . This process was repeated three times to ensure uniformity of the isolated bacteriophage. Purified phages were propagated by inoculating a culture of S . Infantis U1068 at an OD 600 of 0.2 and measuring the absorbance every 30 min until a stable decrease in optical density was achieved. The lysates were centrifuged to eliminate bacteria, chloroform was added at 1% final concentration to the supernatant, and the lysates were stored at 4°C. A sample of each lysate was titrated by the duplex technique, and the concentration in PFU/ml was recorded. DNA Extraction and Genomic Sequencing DNA Extraction and Genomic Sequencing Phage DNA was extracted using the phenol-chloroform method, as previously described (Center for Phage Technology, 2018). A 20 ml volume of high-concentration phage lysate (10 8 -10 9 pfu/ml) was centrifuged at 50,000 g for one hour at 4°C in an ultracentrifuge (Sorvall WX 80) using an AH-650 rotor. The pellet was suspended in 450 µl of Buffer SM (50 mm Tris-HCl, pH 7.5; 100 mm NaCl; 8 mm MgSO 4 ). The suspension was treated with DNase and RNase to final concentrations of 0.01 U/µl and 0.05 mg/ml, respectively. The mixture was incubated at 37°C for one hour. The mixture was then treated with EDTA, SDS, and proteinase K, and incubated at 60°C for 1 hour. An equivalent volume of phenol-chloroform-isoamyl (25:24:1) was added to the mixture, and the mixture was centrifuged at 6000 rpm for 5 min. The aqueous phase was collected, an equivalent volume of chloroform was added, and the centrifugation step was repeated. The upper aqueous phase was collected in a tube, and potassium acetate and 100% ethanol were added in a 2.5 ratio. The mixture was incubated at -20°C overnight. The mixture was centrifuged at 13,000 g for 20 min at 4°C. The DNA pellet was washed twice with 70% ethanol. Finally, the DNA was allowed to dry at room temperature, resuspended in ultrapure water, and stored at -20°C. CsCl Gradient Purification Phage purification by CsCl gradient was performed as previously described (Zhao et al., 2019 ), with some modifications. A 400 ml volume of high-concentration phage lysate (10 8 -10 9 pfu/ml) was centrifuged at 50,000 g for 1 h at 4°C in an ultracentrifuge (Sorvall WX 80) with a TH-641 rotor. The pellet was resuspended in 2 ml of Buffer SM (50 mm Tris-HCl, pH 7.5; 100 mm NaCl; 8 mm MgSO 4 ) and centrifuged at 10,000 g for 5 min in a microcentrifuge to remove any remaining debris. The resulting suspension was placed on a CsCl gradient composed of layers with densities of 1.3, 1.5, and 1.7 g/ml. The gradients were centrifuged at 100,000g for 2 h at 4°C in an AH-650 rotor. The bluish band between the 1.5 and 1.7 g/ml layers was collected and titrated using the double-layer method. Host Range Determination To evaluate the host range of the phages, a total of 29 Salmonella isolates collected from Spanish poultry farms were tested with a focus on S . Infantis strains from different years (2013, n = 9; 2018, n = 10; and 2023, n = 10) (Sevilla-Navarro et al. 2024 a) (Table 1 ). Table 1 List of S. Infantis serovars Name Year Serotype Antigenic formulae Minf 1–10 2023 Infantis 6,7: r: 1,5 Minf 11–20 2018 Infantis 6,7: r: 1,5 Minf 21–29 2013 Infantis 6,7: r: l,5 The ability of the phages to infect these 29 strains was assessed using the spot test assay. Bacterial strains were prepared via DLA technique. Briefly, 200 µL of bacterial inoculum in Luria-Bertani (LB acc. Miller) medium (Sharlau, Barcelona, Spain) at an optical density (OD, λ600 nm) of 0.2 (~ 10⁸ CFU/mL) were mixed with 5 mL of semi-solid LB medium (supplemented with 0.6% agar-agar, VWR Chemicals, Barcelona, Spain) and poured onto solid LB agar plates (1.5% agar). The plates were dried under a laminar flow hood for 10 minutes. Subsequently, 10 µL of each bacteriophage were spotted onto the double-layer agar surface. The plates were incubated at 37.5°C for 24 h, after which phage-induced lysis zones were evaluated on the bacterial lawns (Ahmadi et al. 2016 ; Sevilla-Navarro et al. 2020 ). Due to the high degree of phenotypic similarity among the 24 phages, three representatives were selected for detailed genetic and phenotypic characterization. Efficiency of Plating (EOP) Phage lysates that showed a positive spot were plated on each bacterial strain using the DLA at concentrations of 10 9 -10 4 pfu/ml. The plaques obtained were counted, and the efficiency of plating (EOP) (average pfu in the isolation bacteria/average pfu in the host bacteria) of each phage on each host strain was calculated. The EOP of each phage on each host bacteria, relative to the isolation host, was classified according to the following criteria (Mirzaei & Nilsson 2015): high production EOP ≥ 0,5; medium production 0,1 ≤ EOP < 0,5; low production 0,001 < EOP < 0,1 and no production EOP ≤ 0,001. Stability at pH and Temperature Ranges The thermal stability range of the phages was assessed by incubating 1 ml of a 10 7 pfu/ml suspension at different temperatures (4°C, 25°C, 37°C, 50°C, 60°C, 70°C, and 80°C) for one hour. The suspensions were rapidly titrated after incubation using the DLA method. To assess the pH stability, range of the phages, 0.1 ml of 10 8 pfu/ml lysate of each phage was diluted in 0.9 ml of SM buffer was made using laboratory-grade chemicals at different pH (from 3 to 12) and incubated at 37°C for 1 h. The suspensions were rapidly titrated after incubation using the DLA method. The experiments were performed in independent triplicates. One-Step Growth Curve Phage burst size and lag period were determined using the one-step growth curve assay, according to a previously described method (Zhao et al. 2019 ) with some modifications. Phage lysate (0.1 ml) was added to 0.9 ml of S . Infantis U1068 in the mid-exponential phase (OD 600 = 0.5–0.6, approximately 5×10 8 bac/ml) to reach a concentration of 5×10 7 pfu/ml, resulting in an MOI of 0.01. The mixture was incubated for 5 min at 37°C and then centrifuged at 13,000g for 2 min. The pellet was resuspended in 1 ml of TSB (0.2% MgSO 4 ) and then diluted to 10 − 4 in 30 ml of TSB (0.2% MgSO 4 ). The mixture was incubated at 37°C. Samples of 100 µl were taken at times 0, 5, and 10 min, and then every 10 minutes for 60 min. The samples were titrated by the DLA method, recording the concentration of pfu/ml at each point. The latent period corresponded to the time from infection to the first significant increase in pfu/ml in the culture. The burst size was calculated as the average pfu/ml of the last three maximum points of the experiment divided by the average pfu/ml of the latent time. The experiments were performed in triplicate and the data were graphed on a curve of pfu/ml vs . time in minutes. Lytic Activity in Planktonic Culture Phage lytic activity assays in planktonic cultures were performed as previously described (Liu et al., 2020 ), with modifications. Stationary phase S . Infantis U1068 was diluted to OD 600 = 0.1 (approximately 1×10 8 cfu/ml) in TSB, 0.2% MgSO 4 , and mixed with phage lysates, obtaining different MOIs (10, 1, 0.1, and 0.01) and a final volume of 10 ml. A S . Infantis culture at OD 600 = 0.1 and TSB medium (0.2% MgSO 4 ) were used as positive and negative controls, respectively. The optical density of the cultures was measured in a spectrophotometer and recorded every hour for 8 h. Experiments were performed in triplicate, and data were plotted as an OD 600 vs time curve in hours. Transmission Electron Microscopy (TEM) of phages A transmission electron microscope (TEM) (Tecnai G2 Spirit Twin, FEI, Holland) operated at 80 kV and equipped with an Eagle 4k HR camera was used to identify the phages. Samples were dropped on Formvar-carbon 300 × mesh grids, excess water was removed by using a paper filter, then stained using phosphotungstic acid (PTA) at 2% for 10 s. Genomic analysis of the phages Raw sequencing data was processed using FastQC v0.11.9 and fastp v0.20.1 for quality control and adapter removal (Babraham Bioinformatics, n.d.; Chen et al., 2018 ; Russell, 2018 ). The filtered reads were then assembled with SPAdes v4.0.0 (only-assemble mode) using the following k-mers list: 33, 55, 77, 99 and 127(Bankevich et al., 2012 ; Bujak et al., 2022 ). The assembled contigs were analyzed using Geneious Prime v2025.0.3, and for each phage, the contig with the highest coverage and length was selected as the putative genome. BLAST was performed against the nucleotide collection database to find the closely matching phages to assign a preliminary taxonomic classification (Sevilla-Navarro et al. 2024 c). Genome rearrangement was performed manually using Progressive Mauve, guided by the reference sequence (RefSeq) of the type-phage from the Tlsvirus genus (A. C. E. Darling et al. 2004 ; A. E. Darling et al. 2010 ; Sevilla-Navarro et al. 2024 c). To ensure accuracy in defining the genomic termini, PhageTerm was additionally used to validate the start and end positions of each genome (Garneau et al. 2017 ). Read mapping was performed using BBMap.sh v38.84 to evaluate coverage of the trimmed reads against the assembled contigs (Bushnell 2014 ). The assembly was further refined using Pilon v1.20 (Walker et al. 2014 ). With the corrected genomes, a multiple sequence alignment was conducted using ClustalW implemented in Geneious Prime v2025.0.3 (Sievers et al. 2011 ). For structural and functional annotation, Pharokka v1.4.1 was used with default parameters, and each sequenced coding region was associated with a Phrog functional group (Bouras et al., 2023 ). Protein sequences were manually cross-checked with BLASTp searches (Coudert et al. 2023 ; Sultan-Alolama et al. 2023 ). Pharokka and PhageLeads were used to assist in the prediction of therapeutic suitability while Abricate was used to identify antimicrobial resistance and virulence genes (Seemann 2016 ; Yukgehnaish et al. 2022 ). Phage lifestyle prediction was performed using PhaTYP (Shang et al., 2023 ), and the presence of depolymerase enzymes was predicted using the DePolymerase Predictor (DePP, web version 1.0.0) machine learning tool, considering proteins with a probability greater than 90% as potential depolymerases (Magill & Skvortsov 2023 ). The taxonomic classification of the phages was assessed using VIRIDIC v1.1, which calculates pairwise intergenomic similarities and presents the results as a heatmap and similarity matrix (Moraru et al. 2020 ). Thresholds of 95% for species-level and 70% for genus-level classification were applied. The phage Warwickvirus SLUR29 (accession number NC_054895.1), from a different genus within the same family (subfamily Tempevirinae ), was included as an outgroup in both VIRIDIC and phylogenetic analyses. To support these results, a phylogenetic tree was constructed based on the large terminase subunit ( terL ) gene. Nucleotide sequences were aligned using Clustal Omega (Sievers et al. 2011 ), and the tree was inferred using IQ-TREE v1.6.12 under the maximum likelihood method with 1000 bootstrap replicates (Nguyen et al. 2015 ). The tree was visualized using iTOL v6 (Letunic & Bork 2021 ) and included the same outgroup to root the topology. In addition, a structural comparison was performed using Clinker (Gilchrist & Chooi 2021 ),including representative genomes from the same genus infecting Escherichia coli and Citrobacter . RESULTS Bacteriophage Isolation Three phages were isolated from samples of chicken feces collected at poultry farms in the region of Guayas (Ecuador). All samples were collected on different days. These 3 phages had lytic activity on Salmonella Infantis U1068s and commercial Salmonella Infantis ATCC 5174, Salmonella enterica Serovar Enteritidis ATCC 13076 and no lytic activity on Escherichia coli DH5α. Using S . Infantis U1068s as the host, the lytic halo diameters (mean ± SD, n = 10 replicates) were: GS71: 2.9 ± 0.22 mm; GS156: 2.2 ± 0.27 mm and GS166: 2.6 ± 0.22 mm (Fig. 1 ). Phage Morphology The purified phages were analyzed by transmission electron microscopy (TEM) and classified according to Ackermann's criterion (Ackermann 2007 ). Micrographs revealed that all three phages possessed an icosahedral capsid and a long, non-contractile tail. They belonged to the Caudovirales order and displayed a Siphovirus-like morphology (Fig. 1 ). Host Range The lytic range of the three phages was determined on two commercial strains of S. enterica and one of E. coli . All three phages produced turbid lysis on the commercial Salmonella strains according to the spot test. However, the EOP analysis revealed that none of the phages were able to form plaques on these commercial strains, except for the isolation host. None of the phages showed lytic activity against the E. coli strain. Concerning the field S . Infantis isolated from Spanish poultry farms a total of 87 assays were conducted with phages GS71, GS156, and GS166. Productive infection was defined by the presence of a measurable EOP value, indicating both bacterial susceptibility and effective phage replication. Out of the 87 phage-strain combinations tested, 61 (70.1%) resulted in detectable EOP values. Based on this, 23 of the 29 strains (79.3%) were susceptible to at least one of the phages. Phage GS71 infected 23 strains, with EOP values ranging from 0.71 to 1.00. GS166 infected 22 strains, with EOP values between 0.76 and 1.00. GS156 showed the narrowest host range, infecting 16 strains, with EOP values ranging from 0.76 to 0.94. In all cases, most productive infections yielded EOPs greater than 0.85, indicating high replication efficiency in the susceptible S. Infantis isolates (Fig. 2 ) One-Step Growth Curves A one-step growth curve assay was performed to determine the lag time and burst size of the isolated phages. All three phages had short lag times: 5 minutes for GS71 and GS166, and 10 minutes for GS156. The burst sizes of GS71, GS156, and GS166 were 231, 205, and 215 PFU/cell, respectively (Fig. 3 ). Genomic analysis of the phages The complete genomes of phages GS71, GS156, and GS166 measured 48,587 bp, 48,552 bp, and 48,069 bp, respectively, with a GC content ranging from 41.7–41.8%. Annotation predicted 79 coding sequences (CDS) in both GS71 and GS156, and 76 in GS166. No tRNA genes or pseudogenes were identified (Table 2 ). Table 2 Genomic and taxonomic characteristics of isolated phage genomes. Characterization Phage_GS71 Phage_GS156 Phage_GS166 Size (bp) 48587 48552 48069 GC content (%) 41,8 41,7 41,8 Coding sequences (CDS) 79 79 76 tRNA and pseudogenes 0 0 0 Most similar by Blast Name Accession number: MN994500.1 Phage NBSal001, complete genome Accession number: PP503414.1 Salmonella phage Sephi301i, complete genome Accession number: PP503414.1 Salmonella phage Sephi301i, complete genome Length (bp) 50922 48674 48162 Coverage (%) 83,00 99,00 83,00 E-value 0.0 0.0 0.0 Identity (%) 98,09 98,08 97,7 Predicted Taxonomy Class Caudoviricetes Caudoviricetes Caudoviricetes Family Drexlerviridae Drexlerviridae Drexlerviridae Genus Tlsvirus Tlsvirus Tlsvirus Gene content across the three genomes followed a conserved modular structure, with genes grouped into distinct regions involved in replication, structural assembly, lysis, and packaging. Pharokka identified tail-related proteins in all three phages, and DePP detected one candidate depolymerase per genome, each with a probability greater than 90% (Fig. 4 ). The gene was located on the positive strand in all cases, with slight positional variation: ORF44 in GS71 (23,637–27,497 bp), ORF49 in GS156 (24,951–28,727 bp), and ORF43 in GS166 (23,621–27,481 bp). PhageTerm analysis confirmed that all three phages utilize a Headful (pac) type 1 genome packaging mechanism (Table 3 ). Table 3 Predicted depolymerases in the phage genomes identified by DePP, including packaging modes determined by PhageTerm. Phage ORF START END STRAND FUNCTION PRODUCT PHAGE PACKAGING GS71 44 23637 27497 + tail central tail fiber J HeadFul (pac) type 1 GS156 49 24951 28727 + tail tail protein HeadFul (pac) type 1 GS166 43 23621 27481 + tail tail protein HeadFul (pac) type 1 Additionally, PhageLeads and PhaTYP analyses predicted that all phages follow a strictly lytic infection cycle, with no evidence of temperate markers or virulence factors. Furthermore, ABRicate screening confirmed the absence of antimicrobial resistance genes across all three genomes, reinforcing their suitability for therapeutic or biocontrol applications. VIRIDIC analysis showed that phages GS71 and GS166 shared 98.7% intergenomic similarity. GS156 exhibited slightly lower similarity values with GS71 (92.4%) and GS166 (91.6%). All three phages showed values above the 70% genus-level threshold when compared to reference members of the Tlsvirus genus. In contrast, the outgroup phage SLUR29 displayed values below 75%, confirming its genomic divergence from the studied phages (Fig. 5 ). The phylogenetic tree clustered GS71, GS156, and GS166 into a single monophyletic clade within the Tlsvirus genus (Fig. 6 ). These phages are grouped closely with the type of phage TLS, whereas SLUR29 was placed in a distant branch, consistent with its classification in a different genus of the subfamily Tempevirinae . Comparative genome alignment using Clinker revealed conserved synteny across the three phage genomes. Structural, replication, lysis, and packaging modules were preserved across all isolates. Annotated conserved genes included terminase, tail fiber, DNA polymerase, and lysozyme, among others. Sequence variability was mainly located in short ORFs or regions encoding hypothetical proteins (Fig. 7 ). DISCUSSION The emergence of antibiotic-resistant Salmonella strains has led to the need for alternatives. The increasing prevalence of multidrug-resistant S . Infantis in poultry and food production has renewed interest in bacteriophages as targeted biocontrol agents (Mejía et al . 2020b). In this study, we characterized three lytic phages: GS71, GS156, and GS166, isolated in Ecuador. All three showed lytic activity against local S . Infantis strains collected over multiple years and from diverse sources and locations. S. Infantis is the most common serovar isolated on chicken farms and in retail chains, as well as in human infections in Ecuador. In Ecuador, S. Infantis strains have low genomic divergence (Mejía et al. 2020b). Despite being independently isolated, the phages exhibited high genomic similarity, with intergenomic identities ranging from 91.9–98.7%. This likely reflects the low genomic divergence reported among S. Infantis isolates in Ecuador (Mejía et al. 2020a ) and aligns with studies showing conserved phage populations in ecologically stable host communities(Pyenson et al., 2024 ). Perhaps for this reason, the three phages exhibit high genetic similarity despite all of them being isolated at different times and in different locations. This phenomenon has been previously observed in stable bacterial communities (Pyenson et al. 2024 ). According to current ICTV species demarcation criteria (95% identity), GS71 and GS156 qualify as distinct new species, while GS166 (98.7% similar to GS71) falls within the same species as GS71. VIRIDIC and phylogenetic analysis of the terL gene placed all three phages within a monophyletic cluster of the Tlsvirus genus, supported by high sequence similarity to known members and clear distinction from the outgroup phage SLUR29. Whole-genome alignments using MAFFT revealed strong nucleotide conservation in central regions, while the 5′ and 3′ termini were more variable, containing short ORFs or hypothetical proteins likely reflecting modular rearrangements typical of tailed phages. Comparative synteny analysis with Clinker showed conserved gene modules associated with DNA replication, morphogenesis, lysis, and transcriptional regulation, similar to phages infecting Citrobacter and E. coli (Crossland et al. 2019 ; German & Misra 2001 ; Piya et al. 2019 ; Shaw et al. 2015 ). All three have a gene that codes for a depolymerase responsible for the potent halo around the lysis halo. Functionally, all three phages encode a putative depolymerase gene within their tail modules, predicted with high confidence by DePP and annotated as tail spike or fiber proteins. These enzymes likely accounted for the halo observed around plaques, a hallmark of capsular degradation. Such enzymes are associated with enhanced activity against biofilms and increased penetration of mucosal surfaces (Hua et al. 2022 ; Pan et al. 2019 ; Pires et al. 2016 ). This phenotype was consistent across all three phages in vitro. This depolymerase activity has a superior efficacy against biofilms (Pires et al. 2016 ). They also have small capsids that could facilitate more efficient environmental dispersal and help them to be more effective against biofilms. Notably, GS156 exhibited a narrower host range and lower efficiency of plating (EOP) compared to GS71 and GS166. While GS71 and GS166 lysed 23 and 22 S. Infantis strains respectively (EOP > 0.85), GS156 lysed only 16 strains with reduced efficiency. These included poultry isolate from both Ecuador and Spain, suggesting GS156 retains infectivity across geographic lineages. Its reduced host range may stem from subtle genetic differences or the presence of a unique genomic insertion encoding a polynucleotide kinase (PNK), absent in GS71 and GS166. This enzyme is involved in nucleic acid metabolism and may help phages evade host defenses (Majkowska-Skrobek et al. 2016 ) but could also impose metabolic costs that reduce infectivity. Morphologically, TEM revealed icosahedral capsids (approximately 55 nm) and long, non-contractile tails, consistent with the siphovirus morphotype in Caudoviricetes. While phages with smaller capsids (e.g., podoviruses) may diffuse more efficiently in biofilms or mucus, siphoviruses can still exhibit anti-biofilm activity, especially when carrying depolymerases (Knecht et al. 2020 ; Pires et al. 2016 ). The strong plaque halos support their potential to degrade extracellular matrices. Of the three sequences, two represent new species (GS71 and GS156), while GS166 has a genomic similarity greater than 98.0% with respect to GS71. Therefore, we can only assign two new species, and the phage with the highest similarity to the previous one will retain its original "organism_name" but will be classified within the first described species. Although bacteriophages GS71 and GS166 share 98.7% genetic similarity, they exhibit a 7% variation in their one-step growth curves. This difference may be explained by minimal variations in promoters or regulatory sequences, which could affect replication timing, assembly efficiency, or lysis dynamics. Additionally, mutations in endolysin, holin, or other enzymes involved in phage release may alter burst size or lysis time. Differences in host interaction or genomic rearrangements were ruled out, as both phages display similar host range profiles and no such genomic differences were observed All three phages lack tRNA which is also a characteristic of phages with small genomes. No temperate markers, virulence genes, or antibiotic resistance determinants were found in the genomes of GS71, GS156, or GS166, confirming their strictly lytic nature and genomic safety—key prerequisites for biocontrol applications (Gordillo Altamirano & Barr 2019 ; Song et al. 2024 ; Würstle et al. 2022 ). The genomes also lacked tRNA genes, a common feature in small lytic phages that rely on the host’s translational machinery and are considered suitable for synthetic biology or production platforms (Lomeli-Ortega & Balcázar, 2024 ).Their small size makes them suitable for decreasing their encapsidation capacity and increasing their assembly efficiency in cell-free production systems. This characteristic may be useful in future biotechnological applications (German et al. 2024 ). The lack of tRNA is associated to phages with a host-dependent on transcription machinery (Morgado & Vicente 2019 ). The Tlsvirus genus is the only known phage genera to utilize TolC, an antibiotic and toxin secretory channel protein, as a coreceptor, along with lipopolysaccharide. Tlsviruses attach to the TolC membrane protein and lipopolysaccharide. This dual-receptor strategy implies that if evolutionary dynamics are not considered, treatments with phages or bacterial toxins may lead to resistance(German & Misra 2001 ; Tamer et al. 2021 ). Indeed, experiments have shown that E. coli can rapidly evolve TolC mutations that confer resistance to both colicin and TLS phages, highlighting the need to anticipate resistance when designing phage-based therapies. The three bacteriophages isolated in this study have characteristics that make them suitable for use in cocktails on chicken farms and in retail chains as they are small size, exclusively lytic, carriers of a depolymerase gen and they have a robust genomic safety profile with no antibiotic resistance genes or virulent genetic elements. Taken together, the phages characterized in this study exhibit genomic coherence, safe lytic profiles, biofilm-degrading potential, and host specificity relevant to the Ecuadorian S . Infantis population, supporting their candidacy for application in poultry-associated phage biocontrol strategies. Declarations Author´s Contributions SSN and EFM were responsible for conceptualization. IGC performed the bioinformatic analysis. ICB collected samples and performed biological analysis. SSN, EFM, IGC and ICB Drafting of manuscript. SB Critical revision. Project administration was conducted by ICB, EFM. All authors have read and agreed to the published version of the article. Author Disclosure Statement No competing financial interests exist. Funding Information This study is supported by UEES project 2022-MED-001 titled in Spanish: " Estudio de la actividad de bacteriófagos y de nuevos derivados contra biofilms de Salmonella " Author Contribution SSN and EFM were responsible for conceptualization. IGC performed the bioinformatic analysis. AD performed the electron microscopy analysis. ICB collected samples and performed biological analysis. SSN, EFM, IGC and ICB Drafting of manuscript. SB Critical revision. Project administration was conducted by ICB, EFM. Acknowledgement The authors are grateful to the Dirección de Investigación of Universidad Espíritu Santo and Laboratorio de Caracterización de Nanomateriales of Universidad de las Fuerzas Armadas. We thank Christian Vinueza of the Unidad de Investigación de Enfermedades Transmitidas por Alimentos y Resistencia a los Antimicrobianos (UNIETAR) for the Salmonella Infantis strain isolated in Ecuador. 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A. Morphology of the plaques of phages GS71, GS156, and GS166 produced on a lawn of S. Infantis. B. Micrographs of the phages stained with 2% PTA.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/7b722621f282349f51edc026.png"},{"id":91864329,"identity":"b24831f9-226a-442b-a549-22616b83f2cc","added_by":"auto","created_at":"2025-09-22 13:04:16","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":139096,"visible":true,"origin":"","legend":"\u003cp\u003eHost range and efficiency of plating (EOP) of phages GS71, GS156, and GS166 against 29 \u003cem\u003eSalmonella \u003c/em\u003eInfantis field strains and their propagation strain (PS). EOP was calculated relative to the phage titer obtained on its own PS. Strains are grouped by year of isolation (2023, 2018, 2013), and grey cells indicate absence of productive infection (EOP = 0). Blue intensity reflects increasing EOP values, with a maximum of 1. The propagation strain (PS) was used as the reference host for normalization.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/043dc30766b77feb382e59df.jpeg"},{"id":91864327,"identity":"28a3882e-9a29-4a97-a5bb-933fb14ebba1","added_by":"auto","created_at":"2025-09-22 13:04:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":142984,"visible":true,"origin":"","legend":"\u003cp\u003eOne-step growth curves of phages GS71, GS156, and GS166. Data presented are the average of three independent experiments, along with the corresponding standard deviation. X-axis time in minutes, Y-axis pfu/ml.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/dfa85f29231b556d221134dc.png"},{"id":91865767,"identity":"c6d248ac-6d06-40e3-bdbc-fa9d8d9e613a","added_by":"auto","created_at":"2025-09-22 13:12:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":208446,"visible":true,"origin":"","legend":"\u003cp\u003eWhole-genome alignment of phages. (1) GS156, (2) GS71, and (3) GS166. Generated using Geneious Prime 2024.0.7. The upper panel shows pairwise sequence identity across the aligned genomes, with drops in the curve indicating regions of variability. Predicted coding sequences, identified by Pharokka, are represented as coloured arrows based on functional groups as defined by PHROG. Alignment was performed using MAFFT v1.4.0.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/0db0093b9743d199044c7ef9.png"},{"id":91865766,"identity":"fb81813b-ce90-4a28-9072-1d0b13e679e7","added_by":"auto","created_at":"2025-09-22 13:12:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":186667,"visible":true,"origin":"","legend":"\u003cp\u003eIntergenomic similarity heatmap generated by VIRIDIC. The matrix shows pairwise genomic similarity between the studied phages and reference genomes. Values above 70% indicate genus-level relatedness.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/af25a1d49eb428b8c9d30304.png"},{"id":91864330,"identity":"e8050579-dab4-416f-89d2-a66e5257e01e","added_by":"auto","created_at":"2025-09-22 13:04:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":49054,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree based on \u003cem\u003eterL\u003c/em\u003e gene sequences of phages GS71, GS156, and GS166, along with reference phages. The tree was constructed using the maximum likelihood method, rooted at the midpoint, and visualized with iTOL v.6. Branches corresponding to phages of the genus \u003cem\u003eTlsvirus\u003c/em\u003e are highlighted in red. The outgroup phage (SLUR29) is shown in blue.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/6971e935f5bdf5a942f777c1.png"},{"id":91864331,"identity":"5df18c0f-3ca9-4979-8582-8b526f0c9f68","added_by":"auto","created_at":"2025-09-22 13:04:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":409246,"visible":true,"origin":"","legend":"\u003cp\u003eComparative genome alignment of phages GS71, GS166, and GS156 against reference phages TLS, Stevie, and LL5, generated using Clinker. Arrows represent predicted coding regions labeled by function, and the shaded connections indicate gene homology with identity levels given by the gradient scale.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/ecf4e1fc2ebbdd95e0b0ca51.png"},{"id":94125838,"identity":"a49c0175-0201-4681-85f2-01594a176232","added_by":"auto","created_at":"2025-10-22 16:16:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3134085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7411219/v1/a0843ae4-4105-47e0-9623-fc3aa14fbac7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genomic and Functional Characterization of lytic Tlsvirus bacteriophages targeting Salmonella Infantis isolated from poultry farms in Ecuador","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e-related illnesses are a major global problem concern. Non-typhoidal \u003cem\u003eSalmonella\u003c/em\u003e is estimated to cause 99.8\u0026nbsp;million illnesses and 155,000 deaths each year worldwide (Majowicz et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Most human infections result from the consumption of undercooked contaminated food, especially poultry (Antunes et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Studies have shown that, in Ecuador, \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Infantis (\u003cem\u003eS\u003c/em\u003e. Infantis) is the most prevalent serovar in poultry farms, food, and humans, and it also exhibits multiple patterns of antibiotic resistance (Mej\u0026iacute;a \u003cem\u003eet al.\u003c/em\u003e 2020). A similar trend is observed in other regions, such as Europe, where \u003cem\u003eS.\u003c/em\u003e Infantis is among the top five European Union-acquired serovars involved in human infections. Moreover, it is by far the dominant serovar in the \"broilers\u0026ndash;broiler meat\" category and ranks among the top four serovars across all considered food-animal sources (EFSA 2024).\u003c/p\u003e\u003cp\u003eBacteriophages (phages) are viruses that infect bacteria and possess several characteristics, such as high host specificity, the ability to replicate rapidly within bacterial cells, and the capacity to lyse their host upon completing their replication cycle. They are widely distributed in nature, particularly in environments rich in bacteria such as soil, water, and the human gut. Due to their specificity and bactericidal activity, phages have gained attention as potential alternatives to antibiotics, especially in the context of increasing antimicrobial resistance.\u003c/p\u003e\u003cp\u003eInterest in phages as an alternative to antibiotics on farms has skyrocketed since the FDA approved the first bacteriophage product (phage) for food processing plants in 2006, and the EU approved anti-\u003cem\u003eSalmonella\u003c/em\u003e phages for use in meat products in 2011. The criteria for selecting phages for application in agricultural facilities is that they do not have a lysogenic cycle, carry antibiotic resistance genes, or have pathogenic genes. Considering that 99% of bacteria persist in nature in biofilms(Yan \u0026amp; Bassler \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) a desirable characteristic is that the phages carry depolymerases capable of attacking the biofilms that shelter and protect the bacteria. In this way, the phages, in addition to attacking bacteria, will eliminate them from the biofilms that act as reservoirs.\u003c/p\u003e\u003cp\u003eIn recent decades, there has been a notable increase in the prevalence of multidrug-resistant \u003cem\u003eSalmonella\u003c/em\u003e worldwide, with \u003cem\u003eS.\u003c/em\u003e Infantis emerging as one of the most concerning serovars (Mattock et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this context, phage therapy represents an alternative to antibiotics for controlling \u003cem\u003eSalmonella\u003c/em\u003e proliferation and preventing human infections (Khan \u0026amp; Rahman \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Further, their ability to adapt alongside bacterial evolution makes them especially valuable in dynamic farm environments (Cui et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Limited number of studies have been published regarding the use of bacteriophages in controlling \u003cem\u003eSalmonella\u003c/em\u003e Infantis, yet their potential as biocontrol agents is promising (Battistelli et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Sevilla-Navarro et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003eb; Sevilla-Navarro et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003ec). However, due to the genetic plasticity and high dissemination potential of this bacterium, as well as the presence of different \u003cem\u003eS.\u003c/em\u003e Infantis clades circulating globally, it is essential to have previously isolated and well-characterized bacteriophages that are effective against region-specific strains. This would enhance the efficacy of phage-based interventions and support their targeted application in different epidemiological contexts.\u003c/p\u003e\u003cp\u003eBeyond basic phenotypic characterization, understanding the genomic relationships between candidate phages is critical. Comparative genomics, phylogenetic analyses, and synteny mapping provide insights into their evolutionary stability, functional modules, and potential host-range determinants (Sattar 2022). Moreover, because bacterial resistance to phages can emerge through coevolutionary mechanisms, it is important to anticipate such dynamics when evaluating long-term efficacy (Oechslin \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn this context, the purpose of this study was to isolate and comprehensively characterize lytic phages targeting a \u003cem\u003eSalmonella\u003c/em\u003e Infantis strain isolated in Ecuador, evaluating their biological properties, genomic safety, anti-biofilm potential, and phylogenetic relationships, for potential use as biocontrol agents in food supply chain\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial Strains and Culture Conditions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eS.\u003c/em\u003e Infantis U1068 was isolated in Quito, Ecuador, from chicken carcasses and used as a host for bacteriophage isolation. Additionally, strains of \u003cem\u003eS.\u003c/em\u003e Infantis ATCC 51741, \u003cem\u003eSalmonella enterica\u003c/em\u003e serovar Enteritidis (\u003cem\u003eS\u003c/em\u003e. Enteritidis) ATCC 13076, and \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α were utilized to visualize the morphology of the plaques of phages. Bacteria were incubated until exponential phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;0.6); 100 \u0026micro;l of \u003cem\u003eSalmonella\u003c/em\u003e was added to 100 \u0026micro;l of lysate at 10\u003csup\u003e3\u003c/sup\u003e pfu/ml. The mixture was incubated at 37\u0026deg;C for 5 minutes and then cultured using the double agar technique. The plaques were incubated at 37\u0026deg;C overnight. All strains were grown in liquid Trypto-Casein Soy Broth (TSB) medium (Difco\u0026trade;, BD, USA) at 37\u0026deg;C and 150 rpm. Cultures on solid medium were performed on TSB plates with 1% agar (Difco\u0026trade;, BD, USA). The \u003cem\u003eS.\u003c/em\u003e Infantis strain used to propagate the phages was grown in liquid TSB medium with 0.2% MgSO\u003csub\u003e4\u003c/sub\u003e (Sigma-Aldrich, USA) at 37\u0026deg;C and 150 rpm.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIsolation and Propagation of Bacteriophages\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eSalmonella\u003c/em\u003e bacteriophages were isolated according to a previously described method (Kosznik-Kwaśnicka et al., 2020) with some modifications. Forty-eight chicken feces and wastewater samples were collected from rural areas of the Ecuadorian coast. The samples were enriched by diluting 3 g or 3 ml of the sample in TSB culture medium containing 0.2% MgSO\u003csub\u003e4\u003c/sub\u003e in a 1/10 ratio. Then, 1 ml of exponential-phase \u003cem\u003eS\u003c/em\u003e. Infantis U1068 (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;0.6) and ceftriaxone (CRO) at a final concentration of 10 ppm were added. The enriched samples were incubated at 37\u0026deg;C and 150 rpm overnight. The samples were centrifuged at 4000 rpm for 30 min at 4\u0026deg;C to separate bacteria and solid residues. The supernatant was filtered through a 0.22 \u0026micro;m syringe filter (Merck Millipore, USA), and chloroform was added at a ratio of 1/100. Ten \u0026micro;l of sample was mixed with 100 \u0026micro;l of \u003cem\u003eS\u003c/em\u003e. Infantis, and the solution was cultured by the double-layer agar (DLA) method, using TSB with 0.2% MgSO\u003csub\u003e4\u003c/sub\u003e and 0.5% agar as the upper semisolid medium. The plates were incubated at 37\u0026deg;C and then examined for the presence of lysis plaques (circular clearing spaces) in the bacterial lawn. An isolated plaque from each sample was pricked with a pipette tip and suspended in 300 \u0026micro;l of TSB, 0.2% MgSO\u003csub\u003e4\u003c/sub\u003e. This process was repeated three times to ensure uniformity of the isolated bacteriophage. Purified phages were propagated by inoculating a culture of \u003cem\u003eS\u003c/em\u003e. Infantis U1068 at an OD\u003csub\u003e600\u003c/sub\u003e of 0.2 and measuring the absorbance every 30 min until a stable decrease in optical density was achieved. The lysates were centrifuged to eliminate bacteria, chloroform was added at 1% final concentration to the supernatant, and the lysates were stored at 4\u0026deg;C. A sample of each lysate was titrated by the duplex technique, and the concentration in PFU/ml was recorded.\u003c/p\u003e\n\u003ch3\u003eDNA Extraction and Genomic Sequencing\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eDNA Extraction and Genomic Sequencing\u003c/div\u003e\u003cp\u003ePhage DNA was extracted using the phenol-chloroform method, as previously described (Center for Phage Technology, 2018). A 20 ml volume of high-concentration phage lysate (10\u003csup\u003e8\u003c/sup\u003e-10\u003csup\u003e9\u003c/sup\u003e pfu/ml) was centrifuged at 50,000 g for one hour at 4\u0026deg;C in an ultracentrifuge (Sorvall WX 80) using an AH-650 rotor. The pellet was suspended in 450 \u0026micro;l of Buffer SM (50 mm Tris-HCl, pH 7.5; 100 mm NaCl; 8 mm MgSO\u003csub\u003e4\u003c/sub\u003e). The suspension was treated with DNase and RNase to final concentrations of 0.01 U/\u0026micro;l and 0.05 mg/ml, respectively. The mixture was incubated at 37\u0026deg;C for one hour. The mixture was then treated with EDTA, SDS, and proteinase K, and incubated at 60\u0026deg;C for 1 hour. An equivalent volume of phenol-chloroform-isoamyl (25:24:1) was added to the mixture, and the mixture was centrifuged at 6000 rpm for 5 min. The aqueous phase was collected, an equivalent volume of chloroform was added, and the centrifugation step was repeated. The upper aqueous phase was collected in a tube, and potassium acetate and 100% ethanol were added in a 2.5 ratio. The mixture was incubated at -20\u0026deg;C overnight. The mixture was centrifuged at 13,000 g for 20 min at 4\u0026deg;C. The DNA pellet was washed twice with 70% ethanol. Finally, the DNA was allowed to dry at room temperature, resuspended in ultrapure water, and stored at -20\u0026deg;C.\u003c/p\u003e\n\u003ch3\u003eCsCl Gradient Purification\u003c/h3\u003e\n\u003cp\u003ePhage purification by CsCl gradient was performed as previously described (Zhao et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), with some modifications. A 400 ml volume of high-concentration phage lysate (10\u003csup\u003e8\u003c/sup\u003e-10\u003csup\u003e9\u003c/sup\u003e pfu/ml) was centrifuged at 50,000 g for 1 h at 4\u0026deg;C in an ultracentrifuge (Sorvall WX 80) with a TH-641 rotor. The pellet was resuspended in 2 ml of Buffer SM (50 mm Tris-HCl, pH 7.5; 100 mm NaCl; 8 mm MgSO\u003csub\u003e4\u003c/sub\u003e) and centrifuged at 10,000 g for 5 min in a microcentrifuge to remove any remaining debris. The resulting suspension was placed on a CsCl gradient composed of layers with densities of 1.3, 1.5, and 1.7 g/ml. The gradients were centrifuged at 100,000g for 2 h at 4\u0026deg;C in an AH-650 rotor. The bluish band between the 1.5 and 1.7 g/ml layers was collected and titrated using the double-layer method.\u003c/p\u003e\n\u003ch3\u003eHost Range Determination\u003c/h3\u003e\n\u003cp\u003eTo evaluate the host range of the phages, a total of 29 \u003cem\u003eSalmonella\u003c/em\u003e isolates collected from Spanish poultry farms were tested with a focus on \u003cem\u003eS\u003c/em\u003e. Infantis strains from different years (2013, n\u0026thinsp;=\u0026thinsp;9; 2018, n\u0026thinsp;=\u0026thinsp;10; and 2023, n\u0026thinsp;=\u0026thinsp;10) (Sevilla-Navarro et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003ea) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eList of \u003cem\u003eS.\u003c/em\u003e Infantis serovars\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSerotype\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAntigenic formulae\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMinf 1\u0026ndash;10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInfantis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,7: r: 1,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMinf 11\u0026ndash;20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInfantis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,7: r: 1,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMinf 21\u0026ndash;29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eInfantis\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6,7: r: l,5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe ability of the phages to infect these 29 strains was assessed using the spot test assay. Bacterial strains were prepared via DLA technique. Briefly, 200 \u0026micro;L of bacterial inoculum in Luria-Bertani (LB acc. Miller) medium (Sharlau, Barcelona, Spain) at an optical density (OD, λ600 nm) of 0.2 (~\u0026thinsp;10⁸ CFU/mL) were mixed with 5 mL of semi-solid LB medium (supplemented with 0.6% agar-agar, VWR Chemicals, Barcelona, Spain) and poured onto solid LB agar plates (1.5% agar). The plates were dried under a laminar flow hood for 10 minutes. Subsequently, 10 \u0026micro;L of each bacteriophage were spotted onto the double-layer agar surface. The plates were incubated at 37.5\u0026deg;C for 24 h, after which phage-induced lysis zones were evaluated on the bacterial lawns (Ahmadi et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sevilla-Navarro et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Due to the high degree of phenotypic similarity among the 24 phages, three representatives were selected for detailed genetic and phenotypic characterization.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eEfficiency of Plating (EOP)\u003c/h2\u003e\u003cp\u003ePhage lysates that showed a positive spot were plated on each bacterial strain using the DLA at concentrations of 10\u003csup\u003e9\u003c/sup\u003e-10\u003csup\u003e4\u003c/sup\u003e pfu/ml. The plaques obtained were counted, and the efficiency of plating (EOP) (average pfu in the isolation bacteria/average pfu in the host bacteria) of each phage on each host strain was calculated. The EOP of each phage on each host bacteria, relative to the isolation host, was classified according to the following criteria (Mirzaei \u0026amp; Nilsson 2015): high production EOP\u0026thinsp;\u0026ge;\u0026thinsp;0,5; medium production 0,1\u0026thinsp;\u0026le;\u0026thinsp;EOP\u0026thinsp;\u0026lt;\u0026thinsp;0,5; low production 0,001\u0026thinsp;\u0026lt;\u0026thinsp;EOP\u0026thinsp;\u0026lt;\u0026thinsp;0,1 and no production EOP\u0026thinsp;\u0026le;\u0026thinsp;0,001.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStability at pH and Temperature Ranges\u003c/h3\u003e\n\u003cp\u003eThe thermal stability range of the phages was assessed by incubating 1 ml of a 10\u003csup\u003e7\u003c/sup\u003e pfu/ml suspension at different temperatures (4\u0026deg;C, 25\u0026deg;C, 37\u0026deg;C, 50\u0026deg;C, 60\u0026deg;C, 70\u0026deg;C, and 80\u0026deg;C) for one hour. The suspensions were rapidly titrated after incubation using the DLA method. To assess the pH stability, range of the phages, 0.1 ml of 10\u003csup\u003e8\u003c/sup\u003e pfu/ml lysate of each phage was diluted in 0.9 ml of SM buffer was made using laboratory-grade chemicals at different pH (from 3 to 12) and incubated at 37\u0026deg;C for 1 h. The suspensions were rapidly titrated after incubation using the DLA method. The experiments were performed in independent triplicates.\u003c/p\u003e\n\u003ch3\u003eOne-Step Growth Curve\u003c/h3\u003e\n\u003cp\u003ePhage burst size and lag period were determined using the one-step growth curve assay, according to a previously described method (Zhao et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) with some modifications. Phage lysate (0.1 ml) was added to 0.9 ml of \u003cem\u003eS\u003c/em\u003e. Infantis U1068 in the mid-exponential phase (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;0.6, approximately 5\u0026times;10\u003csup\u003e8\u003c/sup\u003e bac/ml) to reach a concentration of 5\u0026times;10\u003csup\u003e7\u003c/sup\u003e pfu/ml, resulting in an MOI of 0.01. The mixture was incubated for 5 min at 37\u0026deg;C and then centrifuged at 13,000g for 2 min. The pellet was resuspended in 1 ml of TSB (0.2% MgSO\u003csub\u003e4\u003c/sub\u003e) and then diluted to 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e in 30 ml of TSB (0.2% MgSO\u003csub\u003e4\u003c/sub\u003e). The mixture was incubated at 37\u0026deg;C. Samples of 100 \u0026micro;l were taken at times 0, 5, and 10 min, and then every 10 minutes for 60 min. The samples were titrated by the DLA method, recording the concentration of pfu/ml at each point. The latent period corresponded to the time from infection to the first significant increase in pfu/ml in the culture. The burst size was calculated as the average pfu/ml of the last three maximum points of the experiment divided by the average pfu/ml of the latent time. The experiments were performed in triplicate and the data were graphed on a curve of pfu/ml \u003cem\u003evs\u003c/em\u003e. time in minutes.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eLytic Activity in Planktonic Culture\u003c/h2\u003e\u003cp\u003ePhage lytic activity assays in planktonic cultures were performed as previously described (Liu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with modifications. Stationary phase \u003cem\u003eS\u003c/em\u003e. Infantis U1068 was diluted to OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1 (approximately 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e cfu/ml) in TSB, 0.2% MgSO\u003csub\u003e4\u003c/sub\u003e, and mixed with phage lysates, obtaining different MOIs (10, 1, 0.1, and 0.01) and a final volume of 10 ml. A \u003cem\u003eS\u003c/em\u003e. Infantis culture at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1 and TSB medium (0.2% MgSO\u003csub\u003e4\u003c/sub\u003e) were used as positive and negative controls, respectively. The optical density of the cultures was measured in a spectrophotometer and recorded every hour for 8 h. Experiments were performed in triplicate, and data were plotted as an OD\u003csub\u003e600\u003c/sub\u003e \u003cem\u003evs\u003c/em\u003e time curve in hours.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eTransmission Electron Microscopy (TEM) of phages\u003c/h2\u003e\u003cp\u003eA transmission electron microscope (TEM) (Tecnai G2 Spirit Twin, FEI, Holland) operated at 80 kV and equipped with an Eagle 4k HR camera was used to identify the phages. Samples were dropped on Formvar-carbon 300 \u0026times; mesh grids, excess water was removed by using a paper filter, then stained using phosphotungstic acid (PTA) at 2% for 10 s.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eGenomic analysis of the phages\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eRaw sequencing data was processed using FastQC v0.11.9 and fastp v0.20.1 for quality control and adapter removal (Babraham Bioinformatics, n.d.; Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Russell, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The filtered reads were then assembled with SPAdes v4.0.0 (only-assemble mode) using the following k-mers list: 33, 55, 77, 99 and 127(Bankevich et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Bujak et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The assembled contigs were analyzed using Geneious Prime v2025.0.3, and for each phage, the contig with the highest coverage and length was selected as the putative genome. BLAST was performed against the nucleotide collection database to find the closely matching phages to assign a preliminary taxonomic classification (Sevilla-Navarro et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003ec). Genome rearrangement was performed manually using Progressive Mauve, guided by the reference sequence (RefSeq) of the type-phage from the \u003cem\u003eTlsvirus\u003c/em\u003e genus (A. C. E. Darling et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; A. E. Darling et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Sevilla-Navarro et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2024\u003c/span\u003ec). To ensure accuracy in defining the genomic termini, PhageTerm was additionally used to validate the start and end positions of each genome (Garneau et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRead mapping was performed using BBMap.sh v38.84 to evaluate coverage of the trimmed reads against the assembled contigs (Bushnell \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The assembly was further refined using Pilon v1.20 (Walker et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). With the corrected genomes, a multiple sequence alignment was conducted using ClustalW implemented in Geneious Prime v2025.0.3 (Sievers et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). For structural and functional annotation, Pharokka v1.4.1 was used with default parameters, and each sequenced coding region was associated with a Phrog functional group (Bouras et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Protein sequences were manually cross-checked with BLASTp searches (Coudert et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sultan-Alolama et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePharokka and PhageLeads were used to assist in the prediction of therapeutic suitability while Abricate was used to identify antimicrobial resistance and virulence genes (Seemann \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Yukgehnaish et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Phage lifestyle prediction was performed using PhaTYP (Shang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), and the presence of depolymerase enzymes was predicted using the DePolymerase Predictor (DePP, web version 1.0.0) machine learning tool, considering proteins with a probability greater than 90% as potential depolymerases (Magill \u0026amp; Skvortsov \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe taxonomic classification of the phages was assessed using VIRIDIC v1.1, which calculates pairwise intergenomic similarities and presents the results as a heatmap and similarity matrix (Moraru et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thresholds of 95% for species-level and 70% for genus-level classification were applied. The phage \u003cem\u003eWarwickvirus\u003c/em\u003e SLUR29 (accession number NC_054895.1), from a different genus within the same family (subfamily \u003cem\u003eTempevirinae\u003c/em\u003e), was included as an outgroup in both VIRIDIC and phylogenetic analyses.\u003c/p\u003e\u003cp\u003eTo support these results, a phylogenetic tree was constructed based on the large terminase subunit (\u003cem\u003eterL\u003c/em\u003e) gene. Nucleotide sequences were aligned using Clustal Omega (Sievers et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and the tree was inferred using IQ-TREE v1.6.12 under the maximum likelihood method with 1000 bootstrap replicates (Nguyen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The tree was visualized using iTOL v6 (Letunic \u0026amp; Bork \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and included the same outgroup to root the topology.\u003c/p\u003e\u003cp\u003eIn addition, a structural comparison was performed using Clinker (Gilchrist \u0026amp; Chooi \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e),including representative genomes from the same genus infecting \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eCitrobacter\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eBacteriophage Isolation\u003c/h2\u003e\u003cp\u003eThree phages were isolated from samples of chicken feces collected at poultry farms in the region of Guayas (Ecuador). All samples were collected on different days. These 3 phages had lytic activity on \u003cem\u003eSalmonella\u003c/em\u003e Infantis U1068s and commercial \u003cem\u003eSalmonella\u003c/em\u003e Infantis ATCC 5174, \u003cem\u003eSalmonella enterica\u003c/em\u003e Serovar Enteritidis ATCC 13076 and no lytic activity on \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α. Using \u003cem\u003eS\u003c/em\u003e. Infantis U1068s as the host, the lytic halo diameters (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, n\u0026thinsp;=\u0026thinsp;10 replicates) were: GS71: 2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 mm; GS156: 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 mm and GS166: 2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 mm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003ePhage Morphology\u003c/h2\u003e\u003cp\u003eThe purified phages were analyzed by transmission electron microscopy (TEM) and classified according to Ackermann's criterion (Ackermann \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Micrographs revealed that all three phages possessed an icosahedral capsid and a long, non-contractile tail. They belonged to the Caudovirales order and displayed a Siphovirus-like morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eHost Range\u003c/h2\u003e\u003cp\u003eThe lytic range of the three phages was determined on two commercial strains of \u003cem\u003eS. enterica\u003c/em\u003e and one of \u003cem\u003eE. coli\u003c/em\u003e. All three phages produced turbid lysis on the commercial \u003cem\u003eSalmonella\u003c/em\u003e strains according to the spot test. However, the EOP analysis revealed that none of the phages were able to form plaques on these commercial strains, except for the isolation host. None of the phages showed lytic activity against the \u003cem\u003eE. coli\u003c/em\u003e strain.\u003c/p\u003e\u003cp\u003eConcerning the field \u003cem\u003eS\u003c/em\u003e. Infantis isolated from Spanish poultry farms a total of 87 assays were conducted with phages GS71, GS156, and GS166. Productive infection was defined by the presence of a measurable EOP value, indicating both bacterial susceptibility and effective phage replication. Out of the 87 phage-strain combinations tested, 61 (70.1%) resulted in detectable EOP values. Based on this, 23 of the 29 strains (79.3%) were susceptible to at least one of the phages. Phage GS71 infected 23 strains, with EOP values ranging from 0.71 to 1.00. GS166 infected 22 strains, with EOP values between 0.76 and 1.00. GS156 showed the narrowest host range, infecting 16 strains, with EOP values ranging from 0.76 to 0.94. In all cases, most productive infections yielded EOPs greater than 0.85, indicating high replication efficiency in the susceptible \u003cem\u003eS.\u003c/em\u003e Infantis isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eOne-Step Growth Curves\u003c/h2\u003e\u003cp\u003eA one-step growth curve assay was performed to determine the lag time and burst size of the isolated phages. All three phages had short lag times: 5 minutes for GS71 and GS166, and 10 minutes for GS156. The burst sizes of GS71, GS156, and GS166 were 231, 205, and 215 PFU/cell, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eGenomic analysis of the phages\u003c/h2\u003e\u003cp\u003eThe complete genomes of phages GS71, GS156, and GS166 measured 48,587 bp, 48,552 bp, and 48,069 bp, respectively, with a GC content ranging from 41.7\u0026ndash;41.8%. Annotation predicted 79 coding sequences (CDS) in both GS71 and GS156, and 76 in GS166. No tRNA genes or pseudogenes were identified (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGenomic and taxonomic characteristics of isolated phage genomes.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCharacterization\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhage_GS71\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePhage_GS156\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003ePhage_GS166\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eSize (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e48587\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48552\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e48069\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eGC content (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e41,8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41,7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e41,8\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eCoding sequences (CDS)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e76\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003etRNA and pseudogenes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003e\u003cb\u003eMost similar by Blast\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eName\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAccession number: MN994500.1 Phage NBSal001, complete genome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAccession number: PP503414.1 Salmonella phage Sephi301i, complete genome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAccession number: PP503414.1 Salmonella phage Sephi301i, complete genome\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLength (bp)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50922\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48674\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e48162\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCoverage (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e83,00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e99,00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e83,00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-value\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIdentity (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e98,09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e98,08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e97,7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003ePredicted Taxonomy\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eClass\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eCaudoviricetes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eCaudoviricetes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eCaudoviricetes\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFamily\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eDrexlerviridae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eDrexlerviridae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eDrexlerviridae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGenus\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eTlsvirus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eTlsvirus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eTlsvirus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eGene content across the three genomes followed a conserved modular structure, with genes grouped into distinct regions involved in replication, structural assembly, lysis, and packaging. Pharokka identified tail-related proteins in all three phages, and DePP detected one candidate depolymerase per genome, each with a probability greater than 90% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe gene was located on the positive strand in all cases, with slight positional variation: ORF44 in GS71 (23,637\u0026ndash;27,497 bp), ORF49 in GS156 (24,951\u0026ndash;28,727 bp), and ORF43 in GS166 (23,621\u0026ndash;27,481 bp). PhageTerm analysis confirmed that all three phages utilize a Headful (pac) type 1 genome packaging mechanism (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePredicted depolymerases in the phage genomes identified by DePP, including packaging modes determined by PhageTerm.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePhage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eORF\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSTART\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEND\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSTRAND\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFUNCTION\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003ePRODUCT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003ePHAGE PACKAGING\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGS71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23637\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27497\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003ecentral tail fiber J\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eHeadFul (pac) type 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGS156\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e24951\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e28727\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003etail protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eHeadFul (pac) type 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGS166\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e23621\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e27481\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etail\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003etail protein\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eHeadFul (pac) type 1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAdditionally, PhageLeads and PhaTYP analyses predicted that all phages follow a strictly lytic infection cycle, with no evidence of temperate markers or virulence factors. Furthermore, ABRicate screening confirmed the absence of antimicrobial resistance genes across all three genomes, reinforcing their suitability for therapeutic or biocontrol applications.\u003c/p\u003e\u003cp\u003eVIRIDIC analysis showed that phages GS71 and GS166 shared 98.7% intergenomic similarity. GS156 exhibited slightly lower similarity values with GS71 (92.4%) and GS166 (91.6%). All three phages showed values above the 70% genus-level threshold when compared to reference members of the \u003cem\u003eTlsvirus\u003c/em\u003e genus. In contrast, the outgroup phage SLUR29 displayed values below 75%, confirming its genomic divergence from the studied phages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe phylogenetic tree clustered GS71, GS156, and GS166 into a single monophyletic clade within the \u003cem\u003eTlsvirus\u003c/em\u003e genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These phages are grouped closely with the type of phage TLS, whereas SLUR29 was placed in a distant branch, consistent with its classification in a different genus of the subfamily \u003cem\u003eTempevirinae\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eComparative genome alignment using Clinker revealed conserved synteny across the three phage genomes. Structural, replication, lysis, and packaging modules were preserved across all isolates. Annotated conserved genes included terminase, tail fiber, DNA polymerase, and lysozyme, among others. Sequence variability was mainly located in short ORFs or regions encoding hypothetical proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe emergence of antibiotic-resistant \u003cem\u003eSalmonella\u003c/em\u003e strains has led to the need for alternatives. The increasing prevalence of multidrug-resistant \u003cem\u003eS\u003c/em\u003e. Infantis in poultry and food production has renewed interest in bacteriophages as targeted biocontrol agents (Mej\u0026iacute;a \u003cem\u003eet al\u003c/em\u003e. 2020b). In this study, we characterized three lytic phages: GS71, GS156, and GS166, isolated in Ecuador. All three showed lytic activity against local \u003cem\u003eS\u003c/em\u003e. Infantis strains collected over multiple years and from diverse sources and locations. \u003cem\u003eS.\u003c/em\u003e Infantis is the most common serovar isolated on chicken farms and in retail chains, as well as in human infections in Ecuador. In Ecuador, \u003cem\u003eS.\u003c/em\u003e Infantis strains have low genomic divergence (Mej\u0026iacute;a et al. 2020b). Despite being independently isolated, the phages exhibited high genomic similarity, with intergenomic identities ranging from 91.9\u0026ndash;98.7%. This likely reflects the low genomic divergence reported among S. Infantis isolates in Ecuador (Mej\u0026iacute;a et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020a\u003c/span\u003e) and aligns with studies showing conserved phage populations in ecologically stable host communities(Pyenson et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Perhaps for this reason, the three phages exhibit high genetic similarity despite all of them being isolated at different times and in different locations. This phenomenon has been previously observed in stable bacterial communities (Pyenson et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAccording to current ICTV species demarcation criteria (95% identity), GS71 and GS156 qualify as distinct new species, while GS166 (98.7% similar to GS71) falls within the same species as GS71. VIRIDIC and phylogenetic analysis of the terL gene placed all three phages within a monophyletic cluster of the Tlsvirus genus, supported by high sequence similarity to known members and clear distinction from the outgroup phage SLUR29. Whole-genome alignments using MAFFT revealed strong nucleotide conservation in central regions, while the 5\u0026prime; and 3\u0026prime; termini were more variable, containing short ORFs or hypothetical proteins likely reflecting modular rearrangements typical of tailed phages. Comparative synteny analysis with Clinker showed conserved gene modules associated with DNA replication, morphogenesis, lysis, and transcriptional regulation, similar to phages infecting \u003cem\u003eCitrobacter\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e (Crossland et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; German \u0026amp; Misra \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Piya et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Shaw et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll three have a gene that codes for a depolymerase responsible for the potent halo around the lysis halo. Functionally, all three phages encode a putative depolymerase gene within their tail modules, predicted with high confidence by DePP and annotated as tail spike or fiber proteins. These enzymes likely accounted for the halo observed around plaques, a hallmark of capsular degradation. Such enzymes are associated with enhanced activity against biofilms and increased penetration of mucosal surfaces (Hua et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Pires et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This phenotype was consistent across all three phages in vitro. This depolymerase activity has a superior efficacy against biofilms (Pires et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). They also have small capsids that could facilitate more efficient environmental dispersal and help them to be more effective against biofilms.\u003c/p\u003e\u003cp\u003eNotably, GS156 exhibited a narrower host range and lower efficiency of plating (EOP) compared to GS71 and GS166. While GS71 and GS166 lysed 23 and 22 S. Infantis strains respectively (EOP\u0026thinsp;\u0026gt;\u0026thinsp;0.85), GS156 lysed only 16 strains with reduced efficiency. These included poultry isolate from both Ecuador and Spain, suggesting GS156 retains infectivity across geographic lineages. Its reduced host range may stem from subtle genetic differences or the presence of a unique genomic insertion encoding a polynucleotide kinase (PNK), absent in GS71 and GS166. This enzyme is involved in nucleic acid metabolism and may help phages evade host defenses (Majkowska-Skrobek et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) but could also impose metabolic costs that reduce infectivity.\u003c/p\u003e\u003cp\u003eMorphologically, TEM revealed icosahedral capsids (approximately 55 nm) and long, non-contractile tails, consistent with the siphovirus morphotype in Caudoviricetes. While phages with smaller capsids (e.g., podoviruses) may diffuse more efficiently in biofilms or mucus, siphoviruses can still exhibit anti-biofilm activity, especially when carrying depolymerases (Knecht et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Pires et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The strong plaque halos support their potential to degrade extracellular matrices.\u003c/p\u003e\u003cp\u003eOf the three sequences, two represent new species (GS71 and GS156), while GS166 has a genomic similarity greater than 98.0% with respect to GS71. Therefore, we can only assign two new species, and the phage with the highest similarity to the previous one will retain its original \"organism_name\" but will be classified within the first described species.\u003c/p\u003e\u003cp\u003eAlthough bacteriophages GS71 and GS166 share 98.7% genetic similarity, they exhibit a 7% variation in their one-step growth curves. This difference may be explained by minimal variations in promoters or regulatory sequences, which could affect replication timing, assembly efficiency, or lysis dynamics. Additionally, mutations in endolysin, holin, or other enzymes involved in phage release may alter burst size or lysis time. Differences in host interaction or genomic rearrangements were ruled out, as both phages display similar host range profiles and no such genomic differences were observed\u003c/p\u003e\u003cp\u003eAll three phages lack tRNA which is also a characteristic of phages with small genomes. No temperate markers, virulence genes, or antibiotic resistance determinants were found in the genomes of GS71, GS156, or GS166, confirming their strictly lytic nature and genomic safety\u0026mdash;key prerequisites for biocontrol applications (Gordillo Altamirano \u0026amp; Barr \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Song et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; W\u0026uuml;rstle et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The genomes also lacked tRNA genes, a common feature in small lytic phages that rely on the host\u0026rsquo;s translational machinery and are considered suitable for synthetic biology or production platforms (Lomeli-Ortega \u0026amp; Balc\u0026aacute;zar, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).Their small size makes them suitable for decreasing their encapsidation capacity and increasing their assembly efficiency in cell-free production systems. This characteristic may be useful in future biotechnological applications (German et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The lack of tRNA is associated to phages with a host-dependent on transcription machinery (Morgado \u0026amp; Vicente \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe Tlsvirus genus is the only known phage genera to utilize TolC, an antibiotic and toxin secretory channel protein, as a coreceptor, along with lipopolysaccharide. Tlsviruses attach to the TolC membrane protein and lipopolysaccharide. This dual-receptor strategy implies that if evolutionary dynamics are not considered, treatments with phages or bacterial toxins may lead to resistance(German \u0026amp; Misra \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Tamer et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Indeed, experiments have shown that \u003cem\u003eE. coli\u003c/em\u003e can rapidly evolve TolC mutations that confer resistance to both colicin and TLS phages, highlighting the need to anticipate resistance when designing phage-based therapies.\u003c/p\u003e\u003cp\u003eThe three bacteriophages isolated in this study have characteristics that make them suitable for use in cocktails on chicken farms and in retail chains as they are small size, exclusively lytic, carriers of a depolymerase gen and they have a robust genomic safety profile with no antibiotic resistance genes or virulent genetic elements. Taken together, the phages characterized in this study exhibit genomic coherence, safe lytic profiles, biofilm-degrading potential, and host specificity relevant to the Ecuadorian \u003cem\u003eS\u003c/em\u003e. Infantis population, supporting their candidacy for application in poultry-associated phage biocontrol strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor\u0026acute;s Contributions\u003c/h2\u003e\u003cp\u003eSSN and EFM were responsible for conceptualization. IGC performed the bioinformatic analysis. ICB collected samples and performed biological analysis. SSN, EFM, IGC and ICB Drafting of manuscript. SB Critical revision. Project administration was conducted by ICB, EFM. All authors have read and agreed to the published version of the article.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAuthor Disclosure Statement\u003c/strong\u003e\u003cp\u003eNo competing financial interests exist.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding Information\u003c/h2\u003e\u003cp\u003eThis study is supported by UEES project 2022-MED-001 titled in Spanish: \"\u003cem\u003eEstudio de la actividad de bacteri\u0026oacute;fagos y de nuevos derivados contra biofilms de Salmonella\u003c/em\u003e\"\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eSSN and EFM were responsible for conceptualization. IGC performed the bioinformatic analysis. AD performed the electron microscopy analysis. ICB collected samples and performed biological analysis. SSN, EFM, IGC and ICB Drafting of manuscript. SB Critical revision. Project administration was conducted by ICB, EFM.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors are grateful to the Direcci\u0026oacute;n de Investigaci\u0026oacute;n of Universidad Esp\u0026iacute;ritu Santo and Laboratorio de Caracterizaci\u0026oacute;n de Nanomateriales of Universidad de las Fuerzas Armadas. We thank Christian Vinueza of the Unidad de Investigaci\u0026oacute;n de Enfermedades Transmitidas por Alimentos y Resistencia a los Antimicrobianos (UNIETAR) for the Salmonella Infantis strain isolated in Ecuador.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAckermann HW (2007) 5500 phages examined in the electron microscope. 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Front Microbiol 10:420. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2019.00420\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2019.00420\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salmonella, bacteriophage, depolymerase, biofilm, biocontrol, endolysin","lastPublishedDoi":"10.21203/rs.3.rs-7411219/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7411219/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteria of the \u003cem\u003eSalmonella\u003c/em\u003e genus are 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, \u003cem\u003eSalmonella\u003c/em\u003e 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 \u003cem\u003eSalmonella\u003c/em\u003e 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 tp 50ºC 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 \u003cem\u003eS.\u003c/em\u003e Infantis strains. VIRIDIC and phylogenetic analyses placed them within the \u003cem\u003eTlsvirus\u003c/em\u003e genus, showing conserved gene modules for replication, morphogenesis, and lysis. Putative endolysin and depolymerase genes were identified, supporting their strong anti-biofilm activity observed \u003cem\u003ein vitro\u003c/em\u003e. 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 phage cocktails targeting multidrug-resistant \u003cem\u003eS. Infantis\u003c/em\u003e in poultry production.\u003c/p\u003e","manuscriptTitle":"Genomic and Functional Characterization of lytic Tlsvirus bacteriophages targeting Salmonella Infantis isolated from poultry farms in Ecuador","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-22 13:04:11","doi":"10.21203/rs.3.rs-7411219/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48fcadd2-b8be-44ac-aa4b-1b1f72b7c647","owner":[],"postedDate":"September 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-22T16:08:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-22 13:04:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7411219","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7411219","identity":"rs-7411219","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}