Abstract
Foodborne Salmonella enterica and pathogenic Escherichia coli remain major hazards across the farm-to-fork continuum, motivating targeted bacteriophage biocontrol. We isolated two lytic, contractile-tailed phages in Ajman, UAE, CF01 from camel feces and SW01 from municipal sewage and characterized their host range, replication kinetics, and genomes. SW01 lysed only its Salmonella host, whereas CF01 infected multiple E. coli strains and several Salmonella isolates, with efficiency of plating (EOP) indicating high infectivity on E. coli and variable but generally reduced efficiency on Salmonella. One-step growth assays showed SW01 had short latent period (10–20 min), large burst (8–10 × 103 PFU/infected cell), and plateau 3 × 109 PFU/mL at 70–80 min, with an optimal MOI of 0.01. CF01 displayed a 20–30 min latent period, burst 2–3 × 102, plateau 2 × 108 PFU/mL, and optimal MOI 0.1; adsorption was faster than SW01. Illumina MiSeq/Unicycler assemblies yielded single-contig genomes: CF01, 142,427 bp (43.29% GC); SW01, 84,506 bp (44.74% GC). No rRNA genes, CRISPR arrays, integrase/repressor markers, or antimicrobial-resistance determinants were detected under the applied thresholds. Phylogenomic analysis placed CF01 within Salmonella-infecting myoviruses, whereas SW01 formed a distinct lineage more closely related to Erwinia-infecting phages. These results indicate complementary utility: high productivity and low-MOI efficacy of SW01 favor rapid clearance, while CF01 showed broader host range and fast adsorption support inclusion in cocktails. Strictly lytic genomes and favorable kinetics motivate further development of SW01 and CF01 for food-safety applications.
Introduction
Foodborne pathogens such as Salmonella enterica and pathogenic Escherichia coli (notably the O157:H7 serotype) are among the leading global causes of foodborne illness, associated with outbreaks linked to meat, dairy, and fresh produce1. The World Health Organization has estimated that unsafe food causes 600 million illnesses and 420,000 deaths annually worldwide, highlighting the enormous public health burden2. In the United States alone, major foodborne pathogens account for an estimated 9.16 million illnesses each year3.
The overuse and misuse of antibiotics in agriculture and clinical medicine have accelerated the emergence of multidrug-resistant bacteria, complicating treatment and prevention efforts. This “antibiotic resistance crisis” is now considered one of the greatest threats to global health4. In addition, the use of antibiotics in livestock has increased dramatically worldwide, fueling resistance in zoonotic bacteria5. Compounding the problem, many foodborne bacteria, including Salmonella and E. coli O157:H7, can form biofilms that enhance persistence and protect against sanitizers and antimicrobial interventions6.
These challenges underscore the urgent need for alternative approaches to food safety. Bacteriophages, viruses that specifically infect bacteria, have re-emerged as promising biocontrol agents due to their ability to selectively target pathogenic bacteria while sparing beneficial microbiota7. Phage-based strategies have already been shown to reduce foodborne pathogens in laboratory and food system trials8 and commercial phage products such as Salmonelex™ and SalmoFresh™ are approved for use in controlling Salmonella in food processing environments9. Beyond their antibacterial action, phages have also been shown to penetrate and disrupt bacterial biofilms, offering unique advantages compared with conventional antimicrobials10,11,12. An important consideration for phages in food safety applications is their host range. Many phages are highly specific, infecting only a single bacterial species or even particular strains, which can limit their utility when pathogens comprise diverse serovars or variants. Nevertheless, some polyvalent phages with broad host ranges have been identified that can infect multiple serotypes or even distinct genera of bacteria13,14. For example, phage SFP10 was reported to infect both Salmonella enterica and E. coli O157:H7, demonstrating the potential of polyvalent phages to simultaneously target multiple high-risk foodborne pathogens15.
Another practical factor is phage stability under food processing and storage conditions. For food applications, phages must remain viable during refrigeration, mild heating, or other environmental stresses. Certain robust phages maintain lytic activity across broad pH ranges and at temperatures up to ~ 50–60 °C, while others show resilience to desiccation or spray-drying, enabling their formulation into stable products16. Earlier studies have also shown that phages can be successfully applied to fresh products, meat, and dairy, demonstrating their potential across multiple food matrices17,18. These thermal and environmental tolerances are critical in determining how and when phages can be applied in food systems, whether sprayed on raw products, incorporated into packaging, or delivered as dried preparations. Despite these advances, there remains a need to expand the repertoire of well-characterized food-relevant bacteriophages, particularly those isolated from underexplored ecological niches and evaluated through integrated phenotypic and genomic frameworks. Phages recovered from complex environments such as municipal sewage and animal-associated sources represent rich but still incompletely characterized reservoirs of lytic phages with potential utility in food safety applications. Moreover, comparative studies that systematically evaluate multiple phages infecting closely related hosts using standardized workflows remain limited, constraining evidence-based selection of candidates for phage cocktails or targeted interventions.
Unlike many prior studies that characterize bacteriophages largely in isolation, even when multiple phages are reported, the present work adopts a controlled, side-by-side comparative framework that links phenotypic infection dynamics with genome-level features to support rational evaluation of phage candidates for food safety applications. In this context, the present study focuses on the isolation and in-depth characterization of two strictly lytic, contractile-tailed bacteriophages, CF01 and SW01, recovered from distinct environmental sources and targeting two major foodborne pathogens. CF01 was isolated from camel feces using an Escherichia coli host, whereas SW01 was recovered from municipal sewage using a Salmonella host. By examining both phages side-by-side, this work seeks to move beyond descriptive isolation toward a mechanistic understanding of how differences in adsorption dynamics, replication strategies, and productivity may influence their suitability for applied use.
Virion morphology was determined by transmission electron microscopy (TEM), and genome architecture was resolved using Illumina MiSeq sequencing followed by de novo assembly with Unicycler and annotation with Prokka. Taxonomic placement was confirmed, and functional traits—including host range, adsorption kinetics, optimal multiplicity of infection (MOI), and one-step growth parameters (latent period, burst size, and plateau yield)—were quantitatively assessed.
Specifically, this study aims to (i) provide a comprehensive phenotypic and genomic characterization of CF01 and SW01, with emphasis on genes associated with host recognition, lysis, and DNA packaging; (ii) directly compare their infection dynamics and replication efficiency under standardized conditions; and (iii) assess their potential suitability as building blocks for future phage-based interventions targeting foodborne pathogens.
Applied performance in model food matrices (lettuce, chicken, and meat) is addressed in a separate study by the same authors (Sallam et al., under review). By comparing CF01 and SW01 under a unified experimental and analytical workflow, we delineate complementary strengths—broader infectivity and faster adsorption for CF01 versus notably high productivity at low MOI for SW01— that inform rational cocktail design and support their further development as safe, effective tools for microbiological food safety.
Results
Phage isolation and purification
A total of sixteen bacteriophages were isolated from sewage water collected at Ajman Municipality and camel feces obtained from breeding farms in Ajman, UAE. Initial screening by agar overlay assays revealed clear and discrete plaques, confirming the lytic activity of several isolates against Salmonella enterica and Escherichia coli. Phage titers were determined to quantify active viral particles, with considerable variation observed among isolates. Among these, two representative phages were selected for detailed characterization based on their consistently high titers and distinct plaque morphology: Salmonella phage SW01 (5 × 10¹¹ PFU/mL) and E. coli phage CF01 (8 × 10¹⁰ PFU/mL). These phages were purified through repeated single-plaque isolation and propagated to obtain high-titer stocks for subsequent analyses.
Morphological characterization of phages SW01 and CF01
Plaque morphology analysis showed that phage SW01 produced numerous clear, circular plaques with sharply defined boundaries on Salmonella enterica lawns after 18 h of incubation at 37 °C. The plaques measured approximately 1.5–2.0 mm in diameter and exhibited uniform appearance across replicates, indicating stable and efficient lytic activity. TEM revealed virions with an icosahedral head about 58 nm in diameter and a long, contractile tail measuring 90–95 nm. Both intact and contracted tails were visible, confirming the presence of a contractile-tailed morphology typical of myoviruses (Fig. 1).
Morphological characterization of bacteriophage SW01. (a) Plaque morphology of SW01 on a Salmonella lawn showing clear, well-defined lytic plaques (b–d) TEM images of SW01 displaying an icosahedral head and a contractile tail.
Similarly, CF01 formed large, clear plaques with smooth, well-defined edges on Escherichia coli lawns, demonstrating strong lytic activity and consistent plaque formation across replicates. No turbid or diffuse zones were observed, suggesting absence of lysogenic behavior under the tested conditions. TEM examination showed virions with an icosahedral head approximately 60 nm in diameter and a long, contractile tail of about 95 nm. The observation of both extended and contracted tails supported classification within the myovirus morphotype (Fig. 2).
Morphological characterization of bacteriophage CF01. (a) Plaque morphology of CF01 on an E. coli lawn showing clear lytic plaques. (b-d) TEM image of CF01 displaying an icosahedral head and a contractile tail.
Host range analysis of phages SW01 and CF01
Host range analysis (Table 1) revealed that the two phages differed markedly in their lytic profiles. SW01 showed a narrow spectrum, infecting only its original Salmonella host strain. In contrast, CF01 displayed a broader range, lysing multiple E. coli strains as well as several Salmonella isolates.
Efficiency of plating (EOP)
Efficiency of plating (EOP) analysis revealed that phage CF01 exhibited a broader but efficiency-dependent host range (Table 2). High EOP values were observed against Escherichia coli strains, ranging from 0.1 to 0.625, indicating efficient replication on these hosts. CF01 also displayed moderate infectivity toward Salmonella serovars, with EOP values of 0.5 for Salmonella Copenhagen, 0.3 for Salmonella Typhimurium, and a markedly lower value (0.001) for Salmonella Typhi, reflecting variable but generally reduced replication efficiency compared with E. coli.
In contrast, phage SW01 demonstrated a more restricted host range, showing high infectivity toward Salmonella Copenhagen (EOP = 0.8), while no detectable plaque formation was observed on Escherichia coli, other Salmonella serovars, or the remaining tested bacterial species.
Determination of multiplicity of infection
The optimal multiplicity of infection (MOI) for each phage was determined by infecting host cultures at different MOIs (10, 1, 0.1, 0.01, 0.001) and quantifying phage yields (Fig. 3). SW01 reached its highest titer at a low MOI of 0.01 (~ 3 × 10¹⁰ PFU), consistent with its large burst size and high replication efficiency. By contrast, CF01 produced more moderate yields overall, with maximum titers at an intermediate MOI of 0.1 (~ 1.49 × 10⁹ PFU) and comparable yields at MOI 0.01 (~ 1.31 × 10⁹ PFU). These optimal values were subsequently applied in the one-step growth assays.
MOI determination for phages CF01 and SW01. Bacterial cultures were infected at varying MOIs (10, 1, 0.1, 0.01, and 0.001), and phage yields were quantified as plaque-forming units (PFU/mL). Bars represent the mean phage titer from replicate experiments, and error bars indicate the standard deviation (SD).
Replication parameters from one-step growth assays
One-step growth experiments revealed clear differences in the replication dynamics of phages CF01 and SW01 (Fig. 4). Phage CF01 exhibited a latent period of approximately 20–30 min, followed by a gradual increase in phage titer, reaching a plateau of ~ 2 × 10⁸ PFU mL⁻¹ after 70–80 min. In contrast, SW01 displayed a shorter latent period of 10–20 min and a markedly higher replication efficiency, achieving peak titers of ~ 3 × 10⁹ PFU mL⁻¹ within the same time frame. The burst size of SW01 was substantially greater (~ 8–10 × 10³ PFU per infected cell), representing an approximately 40-fold increase compared with CF01 (~ 2–3 × 10² PFU per infected cell). Overall, both phages followed typical one-step growth kinetics consistent with strictly lytic replication cycles.
One-step growth curves of bacteriophages SW01 and CF01.
One-step growth kinetics of phages SW01 and CF01 were determined by monitoring phage titers over time following infection of the host bacterium. Data are presented as mean ± SD from three independent experiments. Phage titers are expressed as PFU mL⁻¹ on a log₁₀ scale.
Phylogenetic placement of phages SW01 and CF01
Phylogenetic analysis based on conserved phage proteins using IQ-TREE (1,000 bootstrap replicates) was performed using the large terminase subunit (TerL) sequences of phages SW01 (GenBank accession no. PX444443.1) and CF01 (GenBank accession no. PX441839.1), together with homologous TerL proteins retrieved from GenBank (Fig. 5).
Phylogenetic analysis based on the large terminase subunit (TerL) revealed that phage CF01 clustered tightly with Salmonella phage SSBI34 with strong bootstrap support (100%), confirming its close evolutionary relationship within this lineage. In contrast, phage SW01 clustered tightly with Salmonella phage SeKF13, forming a strongly supported clade within Salmonella-infecting phages (bootstrap support = 100%). Although several Erwinia phages were positioned in adjacent branches, likely reflecting the conserved nature of the large terminase subunit (TerL), SW01 did not cluster directly with Erwinia-infecting phages. This phylogenetic placement indicates that SW01 represents a distinct Salmonella-associated lineage rather than an Erwinia-related phage (Fig. 5).
Phylogenetic placement of bacteriophages SW01 and CF01 based on the large terminase subunit (TerL). Maximum-likelihood phylogenetic trees were constructed using TerL amino acid sequences of SW01 and CF01 together with homologous sequences retrieved from GenBank, employing IQ-TREE with 1,000 bootstrap replicates. Bootstrap support values > 70% are shown at the nodes. (a) Phylogenetic position of SW01. (b) Phylogenetic position of CF01.
Genomic characterization and comparative analysis of bacteriophages SW01 and CF01
Genome features and organization
Genome sequencing and annotation revealed that both SW01 and CF01 possess linear double-stranded DNA genomes typical of tailed bacteriophages within the class Caudoviricetes. SW01 has a genome size of 84,506 bp with a GC content of 43.8%, whereas CF01 carries a substantially larger genome of 142,427 bp and a GC content of 44.0%. A total of 137 coding DNA sequences (CDSs) were predicted for SW01, compared to 264 annotated features in CF01. Notably, no tRNA genes were identified in SW01, while 10 tRNA genes were detected in CF01, indicating differences in translational capacity between the two phages (Fig. 6).
Circular genome maps of bacteriophages (a) SW01 and (b) CF01. The maps depict genome organization and annotation, showing predicted coding sequences (CDSs) on the forward and reverse strands, GC content, and GC skew. Functional gene modules associated with DNA replication and processing, structural components (head and tail proteins), DNA packaging, and lysis-related functions are highlighted.
Comparative genomic analysis of SW01 and CF01
Comparative analysis of the SW01 and CF01 genomes (Table 3) revealed pronounced differences in genome size, gene content, and functional organization, despite both phages belonging to the class Caudoviricetes. CF01 exhibits a larger and more complex genome architecture, characterized by an expanded structural gene repertoire and the presence of multiple tRNA genes. In contrast, SW01 displays a more compact genome with a higher proportion of genes associated with DNA replication and nucleotide metabolism.
Differences were also observed in the lysis and DNA packaging modules. SW01 encodes a predicted endolysin, whereas CF01 harbors an Rz-like spanin and lacks clearly annotated endolysin and holin genes. In terms of DNA packaging, CF01 encodes both a large terminase subunit and a portal protein, while SW01 encodes only the large terminase subunit. Despite these differences, neither genome contains genes associated with lysogeny, virulence factors, or antibiotic resistance, indicating a strictly lytic lifestyle for both phages.
In-depth annotation of key functional genes
Structural and morphogenesis genes
Genes encoding structural and morphogenesis proteins were identified in both genomes. SW01 encodes a streamlined set of structural components, including a tail tape measure protein, tail fiber protein, baseplate protein, and tail formation protein GpI (gene product I), consistent with a compact virion architecture. Notably, while head-associated structural proteins were not explicitly annotated in the SW01 genome, their presence is supported by transmission electron microscopy, suggesting that these functions are likely encoded by hypothetical or uncharacterized proteins. In contrast, CF01 harbors a more extensive structural module comprising a major capsid protein, minor head protein, head decoration protein, tail fiber protein, baseplate protein, tail assembly chaperone, and neck protein (Neck1), reflecting a more complex virion structure.
DNA replication and nucleotide metabolism
Both phages encode conserved genes involved in DNA replication and nucleotide metabolism. SW01 harbors multiple replication-associated enzymes, including DNA polymerase, DNA primase/helicase, additional helicase, DNA ligase, ribonucleotide reductase subunits, dNMP kinase, and thymidylate synthase, indicating a relatively autonomous replication module. CF01 also encodes key replication and maintenance proteins, such as DNA polymerase, DNA helicase, DNA ligase, exonuclease, endonuclease (ThyA-related), and a single-stranded DNA-binding protein (SSB), supporting efficient genome replication and stability.
Lysis module
Distinct lysis strategies were identified in the two phages. SW01 encodes a predicted endolysin (lysozyme) responsible for peptidoglycan degradation during the terminal stage of infection. In contrast, CF01 encodes a predicted Rz-like spanin, which is implicated in outer membrane disruption during host cell lysis. Canonical holin genes were not clearly annotated in either genome.
DNA packaging machinery
Genes involved in DNA packaging were detected in both genomes. SW01 encodes a conserved large terminase subunit (TerL), whereas CF01 encodes both a large terminase subunit (TerL) and a predicted portal protein, which together mediate genome cleavage, encapsidation, and translocation into the capsid during virion assembly.
Functional relevance of key genomic features
The functional roles of the annotated genes in SW01 and CF01 are consistent with their observed phage biology. Genes encoding structural and tail-associated proteins (including capsid, tail fiber, baseplate, and neck proteins) underpin virion assembly, structural integrity, and adsorption, which are essential for successful initiation of infection.
The presence of conserved DNA replication and nucleotide metabolism genes (such as DNA polymerase, helicase, primase, ligase, and ribonucleotide reductase subunits) supports efficient intracellular genome amplification, enabling productive lytic replication cycles.
DNA packaging proteins, including the large terminase subunit (TerL) and portal protein (in CF01), provide the molecular machinery required for genome cleavage and encapsidation, ensuring the formation of mature, infectious virions.
Importantly, differences in the lysis modules reflect distinct lytic strategies. SW01 encodes a predicted endolysin responsible for peptidoglycan degradation, whereas CF01 encodes an Rz-like spanin involved in outer membrane disruption. Despite these differences, both strategies ultimately converge on effective host cell lysis and virion release.
Collectively, the coordinated action of these functional gene modules underpins the lytic lifestyle, replication efficiency, and biological performance of both phages, providing a genomic basis for their functional behavior.
Summary of genomic features
The genomes of SW01 and CF01 exhibit conserved modular organization typical of tailed bacteriophages, while also displaying distinct genomic strategies in terms of genome size, structural complexity, lysis mechanisms, and DNA packaging architecture. These differences provide valuable context for understanding the biological organization and functional potential of both phages.
Comparative genomic and ANI analysis
Average Nucleotide Identity (ANI) analysis was performed to assess the genomic relatedness of phages SW01 and CF01 to closely related reference phages (Table 4). Phage SW01 displayed high nucleotide identity values ranging from 94.62% to 98.05% with several Salmonella- and Erwinia-infecting phages, including Salmonella phage SeKF13, Salmonella phage vB_SenM_SB18, and Erwinia phage Roscha Roschal. These high ANI values indicate strong genomic conservation and support the placement of SW01 within a well-defined and conserved evolutionary lineage.
In contrast, phage CF01 exhibited a high ANI value (96.05%) exclusively with Salmonella phage SSBI34, identifying it as the closest related phage at the nucleotide level. Substantially lower ANI values (~ 68–70%) were observed when CF01 was compared with Cronobacter phage CR9, Cronobacter phage PBES 02, and Escherichia phage vB_EcoM-LTH01, indicating pronounced nucleotide-level divergence from these phages. Furthermore, pairwise ANI comparison between SW01 and CF01 yielded no detectable nucleotide similarity (ANI n.d., 0% query and subject coverage), confirming that the two phages represent distinct evolutionary lineages.
Proteomic-based phylogenetic reconstruction was consistent with the ANI results, as SW01 clustered tightly with closely related Salmonella and Erwinia phages, whereas CF01 grouped within a clade of Enterobacterales-infecting phages, with Salmonella phage SSBI34 forming its nearest phylogenetic neighbor. The more distant placement of Cronobacter- and Escherichia-infecting phages within this clade aligns with their lower ANI values. Collectively, the ANI and proteomic phylogenetic analyses provide complementary and coherent evidence supporting the distinct evolutionary positions and taxonomic classification of phages SW01 and CF01.
Phylogenetic placement of phages SW01 and CF01
Phylogenomic reconstruction using ViPTree was performed to determine the evolutionary relationships of the two isolates. The analysis, based on a protein distance metric derived from normalized tBLASTx scores, revealed that both SW01 and CF01 clustered within the Caudoviricetes/T4-like (Straboviridae) group (Fig. 7). SW01 nested among established Salmonella-infecting myoviruses, whereas CF01 occupied a related but distinct branch within the same clade, showing closest affinity to enterobacterial phages. This topology is consistent with their host specificity and TEM-based classification as contractile-tailed phages.
Phage phylogenetic tree. The results of the ViPTree analysis using a protein distance metric based on normalized tBLASTx scores plotted on a log scale. (a) SW01. (b) CF01.
BRIG analysis – SW01
To complement the ANI and phylogenetic analyses and provide a visual assessment of genome-wide similarity, circular genome comparison maps were generated using BLAST Ring Image Generator (BRIG), with phage SW01 used as the reference genome (Fig. 8). The BRIG analysis revealed extensive and highly continuous genome-wide similarity between SW01 and closely related Salmonella phages, particularly Salmonella phage SeKF13 and Salmonella phage vB_SenM_SB18, consistent with the high ANI values observed for these phages. Additional comparisons with Salmonella phage SE5 and Erwinia phage Roscha1 also showed substantial similarity across large portions of the SW01 genome, although with minor discontinuities in some regions. Overall, the BRIG patterns indicate that SW01 shares a conserved genomic backbone with these phages, supporting its placement within a well-defined and evolutionarily conserved lineage. These results are fully consistent with the ANI and proteomic phylogenetic analyses.
Circular genome comparison maps of phage SW01 generated using BLAST Ring Image Generator (BRIG). Phage SW01 was used as the reference genome (inner ring). Pairwise BLASTn comparisons were performed with (a) Erwinia phage Roscha1, (b) Salmonella phage SE5, (c) Salmonella phage SeKF13, and (d) Salmonella phage vB_SenM_SB18. Color intensity indicates the degree of nucleotide sequence identity, with darker regions representing higher similarity.
BRIG analysis – CF01
To further explore genome-wide relatedness, circular genome comparison maps were generated using BLAST Ring Image Generator (BRIG), with CF01 used as the reference genome (Fig. 9). The BRIG plots revealed extensive, nearly continuous genome-wide similarity between CF01 and Salmonella phage SSBI34, with strong coverage across most of the CF01 genome, in agreement with the high ANI value (96.05%) observed for this pair. In contrast, comparisons of CF01 with Cronobacter phage CR9, Cronobacter phage PBES 02, and Escherichia phage vB_EcoM-LTH01 showed only patchy and discontinuous similarity, restricted to discrete genomic segments rather than the entire genome. These patterns indicate that CF01 shares a closely related genomic backbone with SSBI34, whereas Cronobacter- and Escherichia-infecting phages are more distantly related and only retain conservation in limited regions. Taken together with the ANI and proteomic phylogenetic analyses, the BRIG results further support the conclusion that CF01 is most closely related to Salmonella phage SSBI34 and is only distantly related to other Enterobacterales phages.
Circular genome comparison maps of phage CF01 generated using BLAST Ring Image Generator (BRIG). Phage CF01 was used as the reference genome (inner ring). Pairwise BLASTn comparisons were performed with (a) Salmonella phage SSBI34, (b) Escherichia phage vB_EcoM-LTH01, (c) Cronobacter phage PBES 02, and (d) Cronobacter phage CR9. Color intensity reflects the level of nucleotide sequence identity, with darker regions indicating higher similarity.
Discussion
In this study, lytic bacteriophages targeting Salmonella enterica and Escherichia coli were recovered from sewage water and camel feces in Ajman, UAE. Sewage proved to be a particularly productive source, yielding the majority of phages with broad host activity, consistent with earlier studies reporting that wastewater contains diverse enteric phages due to the continuous influx of bacterial hosts19,20,21. In contrast, fecal samples often harbored phages with narrower specificity, a pattern also noted by Toribio-Avedillo et al.22. Similarly, Callaway et al.23 reported that although phages were common in pig manure, lytic activity against Salmonella was comparatively rare. The broader host range typically associated with sewage-derived phages reflects exposure to diverse bacterial communities and environmental pressures favoring polyvalence24,25. This ecological distinction mirrors our isolates: SW01, a strictly Salmonella-specific phage, reflects adaptation to a narrow host niche, whereas CF01 displayed broader infectivity across E. coli and Salmonella.
Equally important to the ecological source was the strategy adopted for phage recovery. Our pipeline included sequential filtration, centrifugation, and enrichment, maximizing recovery from low-titer environmental samples while minimizing contamination. Enrichment protocols have been shown to enhance diversity and yield of lytic E. coli and Salmonella phages from wastewater26,27. In our study, phages were selected for clear spots and plaques, with emphasis on uniformity during purification. Uniform plaque morphology is a key criterion for ensuring recovery of clonal, genetically stable phages28. Taken together, plaque morphology and TEM traits confirm that CF01 and SW01 are strictly lytic, contractile-tailed phages assignable to the Myoviridae morphotype29,30.
Host range assays demonstrated that SW01 was specific to Salmonella enterica, while CF01 infected multiple E. coli strains and some Salmonella. Such variability is often attributed to receptor recognition or bacterial defenses. Phage tail fibers determine adsorption specificity and restrict binding to particular serovars31. Conversely, bacteria may employ superinfection exclusion systems to block DNA entry, as described in Enterobacteriaceae prophages32,33. These dynamics explain CF01’s cross-host lysis versus SW01’s narrow range. The efficiency of plating (EOP) was used to define the effectiveness of bacteriophages against target bacteria. An EOP value of 0.5–1.0 was ranked as high efficiency, values between 0.2 and 0.5 were categorized as medium efficiency, while EOP values ranging from 0.001 to 0.2 were classified as low efficiency, and values below 0.001 were considered inefficient34. According to the results, phage CF01 showed high efficiency against Escherichia coli strains, whereas medium to low efficiency was observed against different Salmonella serovars. In contrast, phage SW01 exhibited high efficiency only toward Salmonella Copenhagen, indicating high host specificity. Low or variable EOP values may be attributed to host resistance mechanisms interfering with intracellular phage development or to inefficient bacteriophage adsorption to host cells35.
The morphological characterization of phages SW01 and CF01 confirmed their strictly lytic nature and classification within the contractile-tailed phages. The clear and well-defined plaques produced by both phages indicate rapid adsorption, efficient infection, and complete lysis of the host cells without the establishment of lysogeny. Such plaque clarity is typically associated with virulent phages that follow the lytic replication cycle, supporting their potential application in phage therapy or biocontrol strategies36. The TEM observations further substantiated these findings, revealing virions with icosahedral heads and long contractile tails characteristic of the Myoviridae morphotype under the class Caudoviricetes29. The presence of both intact and contracted tails in the micrographs reflects active infection states and confirms the mechanical nature of DNA injection used by these phages. The measured head and tail dimensions of SW01 and CF01 are consistent with previously described myoviruses infecting Salmonella and Escherichia coli, respectively, suggesting conserved structural organization among lytic phages within this lineage37,38.
The MOI optimization experiments reinforced differences in growth. SW01 achieved maximal yields at a very low MOI (0.01), reflecting its ability to propagate efficiently even at low phage-to-host ratios. This aligns with its exceptionally high burst size (~ 8–10 × 10³ PFU/cell), which is 40-fold greater than CF01 (~ 2–3 × 10² PFU/cell). Burst sizes for E. coli and Salmonella phages typically range from 150 to 400 PFU/cell39, making SW01 exceptional. Similar high productivity has been reported for Salmonella phage LPST15340. In contrast, CF01’s profile resembles polyvalent phages with moderate yields and longer latent phases13. SW01 retained infectivity at elevated temperatures, consistent with earlier reports that tailed phages remain stable at ~ 50–60°C17. It also exhibited stronger antibiofilm activity than CF01, in agreement with evidence that rapid replication and large burst sizes enhance antibiofilm efficacy10,41.
Whole-genome sequencing confirmed that both phages meet key genomic safety criteria, including the absence of integrases, repressors, or AMR genes42,43. CF01 (142 kb, 43.3% GC) and SW01 (84.5 kb, 44.7% GC) yielded high-quality single-contig assemblies, with CDS numbers typical of large lytic myoviruses. GC contents matched known Salmonella phages PS5 and SFP1015,44. In phylogenomic analyses, both clustered with Salmonella-infecting myoviruses, distinct from Enterobacter and Erwinia phages, consistent with evolutionary channeling of myoviruses to enteric hosts36,45. SW01 showed enrichment of nucleotide metabolism AMGs46,47,48 explaining its rapid replication and large burst size, paralleling productive wastewater phages49. CF01, by contrast, had a canonical lytic genome rich in structural and lysis genes, resembling phage vB_EcoM_JS0950. Neither genome encoded lysogeny genes, consistent with therapeutic candidates like vB_EcoM_TU0151. Their lack of tRNAs also fits the profile of virulent phages dependent on host machinery52.
Proteomic-based phylogenetic analyses, such as ViPTree, are valuable for identifying broad evolutionary relationships among bacteriophages. However, due to the conserved nature of core structural proteins, such analyses may group phages with distinct genomic architectures. In the present study, this limitation was addressed by integrating ANI and genome-wide comparison analyses, which provided higher resolution at the nucleotide level and enabled robust differentiation between closely related phages. While both SW01 and CF01 clustered within the Caudoviricetes/T4-like (Straboviridae) group, they occupied distinct phylogenetic branches, indicating related but non-redundant evolutionary lineages. Similar phylogenetic patterns have been documented for phages Felix O1 and SSU5, which anchor deep branches within the Caudoviricetes while retaining clear host specificity36,45. By analogy, the distinct placement of SW01 and CF01 suggests they may provide complementary activities, with SW01 offering rapid Salmonella-specific clearance and CF01 contributing broader infectivity. This evolutionary distinction supports their combined use in phage cocktails, where inclusion of genetically distinct but functionally compatible phages can broaden activity and reduce resistance emergence.
Together, these findings illustrate distinct yet complementary evolutionary strategies: SW01 favors rapid replication and clearance, while CF01 balances broader host adaptation with moderate productivity. Such complementary features are advantageous in phage cocktail design, where combining phages with diverse replication kinetics and host ranges enhances efficacy and mitigates resistance14,53. SW01’s replication efficiency makes it valuable for targeting Salmonella at low host densities, while CF01’s broader host range supports control of mixed E. coli–Salmonella contamination. This is consistent with evidence that combining phages with fast adsorption and high burst sizes improves biocontrol efficacy8,53 and with prior applications of multi-phage cocktails against Salmonella biofilms54. These complementary traits, combined with genomic safety, underscore the suitability of SW01 and CF01 as candidates for rational cocktail-based interventions in food production environments.
Materials and methods
Bacterial strains and growth conditions
Reference strains of Escherichia coli and Salmonella enterica were used as primary hosts for phage isolation and propagation. Additional clinical and environmental isolates of both species were included to determine host range. All strains were maintained on Luria–Bertani (LB) agar and grown in LB broth at 37 °C with shaking at 180 rpm unless otherwise stated. Overnight cultures were diluted 1:100 in fresh LB broth and grown to mid-log phase (OD₆₀₀ ≈ 0.3–0.4) prior to infection assay55.
Phage isolation and purification
Samples were collected from municipal sewage water and camel feces from breeding farms in Ajman, UAE, and enriched with actively growing host cells. For enrichment, 10 mL of sample was mixed with an equal volume of 2× LB broth and inoculated with 100 µL of overnight bacterial culture. Enrichment cultures were incubated at 37 °C for 18 h with shaking. The cultures were centrifuged (10,000 × g, 10 min, 4 °C), and the resulting suspensions were passed through a sterile 0.22 μm membrane filter to eliminate residual cells and fragments, yielding phage-containing lysates. Detection was performed using the double-layer agar method (LB base agar 1.5%, top agar 0.6% supplemented with 10 mM MgSO₄ and 5 mM CaCl₂), where lysates or dilutions were spotted onto bacterial lawns. Distinct plaques were picked and subjected to at least three rounds of purification by single-plaque isolation until clonal preparations were obtained. High-titer stocks were prepared from plate lysates in SM buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 10 mM MgSO₄, 0.01% gelatin), stored at 4 °C for short-term use, and preserved at − 80 °C with 15% glycerol for long-term storage56.
Plaque morphology and transmission electron microscopy (TEM)
Plaque morphology was examined on double-layer agar plates incubated at 37 °C for 18 h, and plaques were measured for diameter and edge clarity. High-titer stocks were generated from confluent lysates by infecting exponentially growing host cultures, followed by centrifugation and 0.45 μm filtration to recover phages in sterile SM buffer57. For ultrastructural examination, purified phage particles (~ 10¹⁰ PFU/mL) were negatively stained with either 2% uranyl acetate or 2% sodium phosphotungstate, as described by Abdel-Haliem and Askora58. A drop of suspension was placed onto 200-mesh carbon-formvar–coated copper grids, excess liquid was removed with filter paper, and grids were examined with a transmission electron microscope (Hitachi H600A, Hitachi Ltd., Japan) at the appropriate accelerating voltage. Particle dimensions (head diameter, tail length) were measured from digital micrographs.
Host range determination
The lytic activity of individual phages was evaluated using spot assays against a panel of bacterial strains, including Escherichia coli, Salmonella enterica, and representative isolates of other clinically relevant genera (Klebsiella pneumoniae, Acinetobacter baumannii, Enterococcus faecalis, Pseudomonas aeruginosa, and Burkholderia cenocepacia)58. Overnight cultures of each strain were mixed with soft agar overlays and poured onto LB plates. Ten microliters of phage suspensions (10⁸ PFU/mL) were spotted onto the bacterial lawns and incubated overnight at 37 °C. Lysis was recorded qualitatively as present (+) or absent (–).
Efficiency of plating (EOP)
Efficiency of plating (EOP) was determined using an agar overlay assay and performed in triplicate. EOP values were calculated as the ratio of the average plaque-forming units (PFU) obtained on each target bacterial strain to the average PFU obtained on the corresponding reference host strain, following previously described methods59.
Determination of the optimal multiplicity of infection
The optimal multiplicity of infection for each phage was determined by infecting host cultures (Escherichia coli for CF01 and Salmonella enterica for SW01) at different MOIs (0.001, 0.01, 0.1, 1, and 10). After incubation at 37 °C, phage yields were quantified by the double-layer agar method, and the MOI that produced the highest phage titer was considered optimal60.
One-step growth curve analysis
To evaluate the infectivity and replication capacity of the phages, one-step growth curve experiments were performed at the optimal MOI identified for each phage. Mid-log bacterial cultures (OD₆₀₀ ≈ 0.3) were infected at the selected MOI for each phage (SW01: 0.01; CF01: 0.1) and allowed to adsorb for 10 min at 37 °C. Unbound phages were removed by centrifugation (6,000 × g, 5 min) followed by washing with pre-warmed LB broth. The infected cells were resuspended in fresh LB and incubated at 37 °C with shaking. Samples were taken at 10-min intervals over a 120-min period, serially diluted, and plated using the double-layer agar method to determine phage titers (PFU/mL). The latent period was defined as the time from adsorption until the first rise in phage counts, while the burst size was calculated from the increase in PFU during the rise phase relative to the initial number of infected cells. Plateau yield was recorded as the maximum PFU/mL obtained. All experiments were conducted in triplicate61,62.
DNA extraction, sequencing, and bioinformatic analysis
Phage genomic DNA was isolated from high-titer lysates using polyethylene glycol (PEG) precipitation followed by enzymatic digestion and column-based purification with the Qiagen QIAamp DNA Mini Kit, with minor modifications63,64. Briefly, 4–8 mL of phage lysate (free of chloroform) was mixed with 5× phage precipitation solution (20% PEG 8000, 2.5 M NaCl) at a ratio of 1:4 (v/v) and incubated at 4 °C for 2 h to overnight. Precipitated phages were collected by centrifugation (10,000 × g, 30 min, 4 °C) and resuspended in 360 µL of phage resuspension buffer (1 M NaCl, 10 mM Tris-HCl, pH 7.5). To eliminate residual bacterial nucleic acids, the suspension was treated with DNase I (1 U) and RNase A (20 µg) in 1× DNase buffer at 37 °C for 30 min, followed by inactivation with 20 mM EDTA. Proteinase K (20 mg/mL, 1.25 µL) was then added and incubated at 56 °C for 1.5 h to digest the phage capsid.
For DNA purification, 200 µL of the lysed phage suspension was mixed with an equal volume of Buffer AL (Qiagen) and incubated at 70 °C for 10 min, followed by addition of 200 µL absolute ethanol (96–100%). The mixture was loaded onto a QIAamp Mini spin column in multiple steps (≤ 750 µL per load) and washed sequentially with Buffer AW1 (500 µL) and Buffer AW2 (500 µL), with centrifugation steps according to the manufacturer’s instructions unless otherwise specified. Columns were dried at 20,000 × g for 3 min to remove residual ethanol, and DNA was eluted with 30 µL of AE buffer. DNA concentration and purity were assessed by Nanodrop spectrophotometry and Qubit fluorometry, and integrity was verified on 1% agarose gel electrophoresis.
Sequencing libraries were prepared with a commercial library preparation kit and sequenced on an Illumina MiSeq platform. Raw reads in FASTQ format were processed on the Galaxy platform. Quality assessment was performed with FastQC v0.11.965, and de novo assembly was carried out using Unicycler v0.4.866. Genome annotation was conducted with Prokka v1.14.667, followed by functional reannotation with KEGG GhostKOALA68. Homology searches were performed with BLAST against the KEGG GENES database. Open reading frames were predicted with Prodigal v2.6.369 (single-genome mode); tRNAs with Aragorn v1.2.3870; rRNAs with Barrnap v0.971; and CRISPR arrays were identified using MinCED v0.4.2, which implements an optimized version of the CRISPR Recognition Tool (CRT) algorithm72. Phylogenetic relationships were inferred using the ETE3 pipeline73. Circular genome maps were generated with Proksee74.
Comparative genomic analysis
Comparative genomic analyses were conducted to evaluate differences in genome size, GC content, gene content, and functional gene organization between phages SW01 and CF01. Annotated genomes were examined to identify conserved and variable genomic regions, with particular emphasis on genes involved in DNA replication, virion structure, host recognition, and lysis.
Average nucleotide identity (ANI) analysis
Average Nucleotide Identity (ANI) analysis was performed to assess nucleotide-level relatedness between phages SW01 and CF01 and closely related reference phage genomes retrieved from the NCBI GenBank database. Pairwise ANI values were calculated using OrthoANIu with default parameters75. ANI results were used to evaluate genomic similarity and delineate evolutionary relationships among the analyzed phages.
Phylogenomic analysis
Phylogenomic reconstruction was performed using ViPTree to infer evolutionary relationships based on whole-proteome comparisons76. The analysis relies on a proteomic distance matrix derived from normalized tBLASTx scores calculated between query phages and reference viral genomes. Phylogenetic trees were visualized in a circular layout and interpreted in the context of viral taxonomy and host specificity.
Circular genome comparison
Genome-wide similarity was further examined using BLAST Ring Image Generator (BRIG)77. Circular genome comparison maps were generated using phages SW01 or CF01 as reference genomes, with selected closely related phages included for pairwise BLASTn comparisons. Color intensity in the BRIG plots reflects the degree of nucleotide sequence identity across the genome, allowing visualization of conserved and divergent genomic regions.
Conclusions
This study provides a comparative characterization of two strictly lytic, contractile-tailed phages, CF01 and SW01, that meet key genomic safety criteria (no lysogeny markers or AMR genes). CF01 adsorbed rapidly and performed best at an intermediate MOI (0.1) with moderate yields, whereas SW01 achieved markedly higher productivity, shorter latent period, greater plateau titers, and reached its optimum at a low MOI (0.01). Across assays, replication metrics were more predictive of sustained bacterial suppression than adsorption alone. Complementary applied tests (Sallam et al., under review) further indicate superior stability and antibiofilm activity for SW01, underscoring its promise for food-safety use. Together, these results provide a practical framework for rational phage selection: deploy highly productive phages like SW01 to drive rapid clearance, complemented by phages such as CF01 to balance performance characteristics within cocktails. Future work should validate efficacy in food matrices and monitor resistance dynamics.
Data availability
The nucleotide sequences of phages SW01 and CF01 have been deposited in GenBank under accession numbers PX444443.1 (SW01) and PX441839.1 (CF01). All other data generated or analyzed during this study are included in this article.
References
Lee, H. & Yoon, Y. Etiological agents implicated in foodborne illness world wide. Food Sci. Anim. Resour. 41, 1 (2021).
World Health Organization, Investing to overcome the global impact of neglected tropical diseases: third WHO report on neglected tropical diseases Vol. 3. (2015).
Scallan, E. et al. Foodborne illness acquired in the united States—Major pathogens. Emerg. Infect. Dis. 17, 7 (2011).
Shaaban, M. T., Menisy, S. I. & Hegazy, A. M. Microbiological and molecular studies on Salmonella spp. Isolated from broilers in Kafr El-Sheikh governorate, Egypt. J. Biosci. Appl. Res. 1 (2), 59–67 (2015).
Van Boeckel, T. P. et al. Global trends in antimicrobial use in food animals. Proc. Natl. Acad. Sci. U S A. 112, 5649–5654 (2015).
Milho, C. et al. Escherichia coli and Salmonella Enteritidis dual-species biofilms: interspecies interactions and antibiofilm efficacy of phages. Sci. Rep. 9 (19), 1–15 (2019).
Pham-Khanh, N. H. et al. Isolation, characterisation and complete genome sequence of a tequatrovirus Phage, Escherichia phage KIT03, which simultaneously infects Escherichia coli O157:H7 and Salmonella enterica. Curr. Microbiol. 76, 1130–1137 (2019).
Islam, M. S. et al. Application of a phage cocktail for control of Salmonella in foods and reducing biofilms. Viruses 2019. 11, Page 841 (11), 841 (2019).
Soffer, N. et al. Bacteriophages safely reduce Salmonella contamination in pet food and Raw pet food ingredients. Bacteriophage 6, e1220347 (2016).
Gutiérrez, D., Rodríguez-Rubio, L., Martínez, B., Rodríguez, A. & García, P. Bacteriophages as weapons against bacterial biofilms in the food industry. Front. Microbiol. 7, 189912 (2016).
Donlan, R. M. Preventing biofilms of clinically relevant organisms using bacteriophage. Trends Microbiol. 17, 66–72 (2009).
Sillankorva, S., Neubauer, P. & Azeredo, J. Phage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus. Biofouling 26, 567–575 (2010).
Yu, P., Mathieu, J., Li, M., Dai, Z. & Alvarez, P. J. J. Isolation of polyvalent bacteriophages by sequential multiple-host approaches. Appl. Environ. Microbiol. 82, 808–815 (2016).
Hyman, P. & Abedon, S. T. Bacteriophage host range and bacterial resistance. Adv. Appl. Microbiol. 70, 217–248 (2010).
Park, M. et al. Characterization and comparative genomic analysis of a novel bacteriophage, SFP10, simultaneously inhibiting both Salmonella enterica and Escherichia coli O157:H7. Appl. Environ. Microbiol. 78, 58–69 (2012).
Shaaban, M. T., Abosenna, A., Ahmed, A. & Sallam, O. Characterization of Lactobacillus phages isolated from dairy products handled in Egyptian markets. Egypt. J. Exp. Biol. (Bot), 16(2), 231–242(2020).
Moye, Z. D., Woolston, J. & Sulakvelidze, A. Bacteriophage applications for food production and processing. Viruses 2018. 10, Page 205 (10), 205 (2018).
García, P., Martínez, B., Obeso, J. M. & Rodríguez, A. Bacteriophages and their application in food safety. Lett. Appl. Microbiol. 47, 479–485 (2008).
Dias, E., Ebdon, J. & Taylor, H. The application of bacteriophages as novel indicators of viral pathogens in wastewater treatment systems. Water Res. 129, 172–179 (2018).
McMinn, B. R., Ashbolt, N. J. & Korajkic, A. Bacteriophages as indicators of faecal pollution and enteric virus removal. Lett. Appl. Microbiol. 65, 11–26 (2017).
Okoh, A. I., Sibanda, T. & Gusha, S. S. Inadequately treated wastewater as a source of human enteric viruses in the environment. Int. J. Environ. Res. Public. Health. 7, Pages 2620–2637 (7), 2620–2637 (2010).
Toribio-Avedillo, D., Blanch, A. R. & Muniesa, M. Rodríguez-Rubio, L. Bacteriophages as fecal pollution indicators. Viruses 2021. 13, 1089 (2021).
Callaway, T. R. et al. Occurrence of Salmonella-specific bacteriophages in swine feces collected from commercial farms. Foodborne Pathog Dis. 7, 851–856 (2010)
Diez-Gonzalez, F., Akhtar, M. & Viazis, S. Isolation, identification and characterization of lytic, wide host range bacteriophages from waste effluents against Salmonella enterica serovars. Food Control. 38, 67–74 (2014).
De Leeuw, M., Baron, M., Brenner, A. & Kushmaro, A. Genome analysis of a novel broad host range Proteobacteria phage isolated from a bioreactor treating industrial wastewater. Genes 8, 40 (2017).
Beheshti Maal, K., Delfan, A. S. & Salmanizadeh, S. Isolation and identification of two novel Escherichia coli bacteriophages and their application in wastewater treatment and coliform’s phage therapy. Jundishapur J. Microbiol. 8, e14945 (2015).
McLaughlin, M. R., Balaa, M. F., Sims, J. & King, R. Isolation of Salmonella bacteriophages from swine effluent lagoons. J. Environ. Qual. 35, 522–528 (2006).
Tikunova, N. V., Knezevic, P., Glonti, T. & Pirnay, J. P. In vitro techniques and measurements of phage characteristics that are important for phage therapy success. Viruses 2022. 14, 1490 (2022).
Bradley, D. E. Ultrastructure of bacteriophage and bacteriocins. Bacteriol. Rev. 31, 230 (1967).
Ackermann, H. W. Basic phage electron microscopy. Methods Mol. Biol. 501, 113–126 (2009).
Nobrega, F. L. et al. Targeting mechanisms of tailed bacteriophages. Nat. Rev. Microbiol. 16 (12), 760–773 (2018).
Bucher, M. J. et al. Phage against the machine: the SIE-ence of superinfection exclusion. Viruses 2024. 16, 1348 (2024).
Labrie, S. J., Samson, J. E. & Moineau, S. Bacteriophage resistance mechanisms. Nat. Rev. Microbiol. 2010. 8:5 (8), 317–327 (2010).
Mirzaei, M. K. & Nilsson, A. S. Isolation of phages for phage therapy: A comparison of spot test and efficiency of plating analyses for determination of host range and efficacy. PLoS ONE. 10, e0118557 (2015).
Azeredo, J. & Sillankorva, S. Bacteriophage Therapy: from Lab To Clinical Practice (Springer Science, 2018).
Kim, M., Kim, S., Park, B. & Ryu, S. Core lipopolysaccharide-specific phage SSU5 as an auxiliary component of a phage cocktail for Salmonella biocontrol. Appl. Environ. Microbiol. 80, 1026–1034 (2014).
Kropinski, A. M. et al. The host-range, genomics and proteomics of Escherichia coli O157:H7 bacteriophage rV5. Virol. J. 10, 1–12 (2013).
Yang, F., Wang, L., Zhou, J., Xiao, H. & Liu, H. In situ structures of the ultra-long extended and contracted tail of myoviridae phage P1. Viruses 15, 1267 (2023).
López-Cuevas, O., Castro-del Campo, N., León-Félix, J., González-Robles, A. & Chaidez, C. Characterization of bacteriophages with a lytic effect on various Salmonella serotypes and Escherichia coli O157:H7. (2011). https://doi.org/10.1139/w11-099 57, 1042–1051.
Islam, M. S. et al. Characterization of Salmonella phage LPST153 that effectively targets most prevalent Salmonella serovars. Microorganisms 2020. 8, 1089 (2020).
Meneses, L. et al. A systematic review of the use of bacteriophages for in vitro biofilm control. Eur. J. Clin. Microbio Infect. Dis. 42, 919–928 (2023).
Peng, X., Ed-Dra, A. & Yue, M. Whole genome sequencing for the risk assessment of probiotic lactic acid bacteria. Crit. Rev. Food Sci. Nutr. 63, 11244–11262 (2023).
Philipson, C. W. et al. Characterizing phage genomes for therapeutic applications. Viruses 2018. 10, Page 188 (10), 188 (2018).
Duc, H. et al. Isolation, characterization and application of a polyvalent phage capable of controlling Salmonella and Escherichia coli O157: H7 in different food matrices. Food res. int. 131, 108977 (2020).
Whichard, J. M., Sriranganathan, N. & Pierson, F. W. Suppression of Salmonella growth by Wild-Type and Large-Plaque variants of bacteriophage Felix O1 in liquid culture and on chicken Frankfurters. J. Food Prot. 66, 220–225 (2003).
Thompson, L. R. et al. Phage auxiliary metabolic genes and the Redirection of cyanobacterial host carbon metabolism. Proc. Natl. Acad. Sci. U S A. 108, E757–E764 (2011).
Tian, F. et al. Prokaryotic-virus-encoded auxiliary metabolic genes throughout the global oceans. Microbiome 12 (1), 1–16 (2024).
Warwick-Dugdale, J., Buchholz, H. H., Allen, M. J. & Temperton, B. Host-hijacking and planktonic piracy: how phages command the microbial high seas. Virol. J. 16 (1), 1–13 (2019).
Topka, G. et al. Characterization of bacteriophage vB-EcoS-95, isolated from urban sewage and revealing extremely rapid lytic development. Front. Microbiol. 10, 411311 (2019).
Zhou, Y., Bao, H., Zhang, H. & Wang, R. Isolation and characterization of lytic phage vB_EcoM_JS09 against clinically isolated Antibiotic-Resistant avian pathogenic Escherichia coli and enterotoxigenic Escherichia coli. Intervirology 58, 218–231 (2015).
Nepal, R. et al. Genomic characterization of three bacteriophages targeting multidrug resistant clinical isolates of Escherichia, Klebsiella and Salmonella. Arch. Microbiol. 204, 334 (2022).
Bailly-Bechet, M., Vergassola, M. & Rocha, E. Causes for the intriguing presence of tRNAs in phages. Genome Res. 17, 1486–1495 (2007).
Ribeiro, J. M. et al. Comparative analysis of effectiveness for phage cocktail development against multiple Salmonella serovars and its biofilm control activity. Sci. Rep. 13 (1), 1–17 (2023).
Pottker, E. S. et al. Bacteriophages as an alternative for biological control of biofilm-forming Salmonella enterica. Food Sci. Technol. Int. 30, 197–206 (2024).
Hyman, P. & Abedon, S. T. Practical methods for determining phage growth parameters. In Bacteriophages: Methods and protocols, Volume 1: isolation, characterization, and Interactions, eds. Clokie, M. R. J. & Kropinski, A. M., 175–202. Totowa, NJ: Humana (2009).
Van Twest, R. & Kropinski, A. M. Bacteriophage enrichment from water and soil. Methods Mol. Biol. 501, 15–21 (2009).
Jaiswal, S. K. & Dhar, B. Morphology and general characteristics of phages specific to lens culinaris rhizobia. Biol. Fertil. Soils. 46, 681–687 (2010).
Abdel-Haliem, M. E. F. & Askora, A. Isolation and characterization of bacteriophages of Helicobacter pylori isolated from Egypt. Future Virol. 8, 821–826 (2013).
Artawinata, P. C., Lorraine, S. & Waturangi, D. E. Isolation and characterization of bacteriophages from soil against food spoilage and foodborne pathogenic bacteria. Sci. Rep. 13, 9282 (2023).
Pazhouhnia, S., Bouzari, M. & Arbabzadeh-Zavareh, F. Isolation, characterization and complete genome analysis of a novel bacteriophage vB_ EfaS-SRH2 against Enterococcus faecalis isolated from periodontitis patients. Sci. Rep. 12, 13268 (2022).
Kropinski, A. M. Practical advice on the One-Step growth curve. Methods Mol. Bio. 1681, 41–47 (2018).
Bacteriophages Biology and Applications. (2004). https://doi.org/10.1201/9780203491751
Jakočiūnė, D., Moodley, A. A., Rapid Bacteriophage, D. N. A. & Extraction Method. Methods Protocols 2018, Vol. 1 1, 27 (2018).
Burgener, E. B. et al. Methods for extraction and detection of Pf bacteriophage DNA from the sputum of patients with cystic fibrosis. Phage 1 (2), 100–108 (2020).
Andrews, S. FastQC: a quality control tool for high throughput sequence data (2010).
Wick, R. R., Judd, L. M., Gorrie, C. L. & Holt, K. E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13, e1005595 (2017).
Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).
Kanehisa, M., Sato, Y. & Morishima, K. BlastKOALA and ghostkoala: KEGG tools for functional characterization of genome and metagenome sequences. J. Mol. Biol. 428, 726–731 (2016).
Hyatt, D. et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinf. 11 (1), 11–11 (2010). (2010).
Laslett, D. & Canback, B. ARAGORN, a program to detect tRNA genes and TmRNA genes in nucleotide sequences. Nucleic Acids Res. 32, 11–16 (2004).
Seemann, T. Barrnap 0.9: rapid ribosomal RNA prediction (2013). Available online: https://github.com/tseemann/barrnap
Bland, C. et al. CRISPR recognition tool (CRT): a tool for automatic detection of clustered regularly interspaced palindromic repeats. BMC Bioinform. 8, 1–8 (2007).
Huerta-Cepas, J., Serra, F. & Bork, P. ETE 3: Reconstruction, Analysis, and visualization of phylogenomic data. Mol. Biol. Evol. 33, 1635–1638 (2016).
Grant, J. R. et al. Proksee: in-depth characterization and visualization of bacterial genomes. Nucleic Acids Res. 51, W484–W492 (2023).
Yoon, S. H., Ha, S. M., Lim, J., Kwon, S. & Chun, J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 110, 1281–1286 (2017).
Nishimura, Y. et al. ViPTree: the viral proteomic tree server. Bioinformatics 33, 2379–2380 (2017).
Alikhan, N. F., Petty, N. K., Zakour, B., Beatson, S. A. & N. L. & BLAST ring image generator (BRIG): simple prokaryote genome comparisons. BMC Genom. 12, 402 (2011).
Funding
No funding was received for this study.
Author information
Authors and Affiliations
Contributions
O.S. performed all experiments and prepared the initial draft. A.E.-K., S.K.M., and M.T.S. contributed to writing and revising the manuscript. Statistical analyses were performed by A.E.-K. and S.K.M. Chemicals were provided by T.M.O. All authors approved the final manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Shaaban, M.T., Sallam, O., Manikandan, S.K. et al. Comparative morphological, genomic, and functional characterization of two newly isolated lytic caudoviricetes phages targeting Escherichia coli and Salmonella. Sci Rep 16, 10340 (2026). https://doi.org/10.1038/s41598-026-39186-z
Received:
Accepted:
Published:
Version of record:
DOI: https://doi.org/10.1038/s41598-026-39186-z
