Isolation and characterization of a novel lytic bacteriophage Ecolivirus Myo-P293 targeting avian pathogenic Escherichia coli

Abstract

A novel lytic bacteriophage, Ecolivirus Myo‑P293 (P293), targeting avian pathogenic Escherichia coli (APEC), was isolated from duck farm sewage in Jiangsu, China. P293 formed clear plaques approximately 2 mm in diameter and displayed the characteristic morphology of Myoviridae family, with an icosahedral head (~ 70 nm) and a contractile tail (~ 100 nm), as observed under transmission electron microscopy. Adsorption assays showed that over 70% of phages adsorbed to host cells within 5 min. One-step growth analysis revealed a latent period of approximately 30 min and a burst size of 244 ± 36 PFU per infected cell. P293 exhibited stability across a pH range of 5–9 and at temperature between 20 and 40 °C, but its infectivity was significantly reduced when exposed to temperature ≥ 60 °C or 60% ethanol. The phage effectively disrupted mature E. coli biofilms, achieving approximately 45% clearance after 24 h of treatment, and also inhibited biofilm formation in a dose-dependent manner. Whole-genome sequencing of P293 revealed an 89.5 kb double-stranded DNA genome encoding 95 open reading frames (ORFs), including modules related to structure, replication, lysis, and host interaction. Approximately 40% of the encoded genes are annotated as hypothetical proteins. Phylogenetic and comparative genomic analyses placed P293 within the unclassified Felixounavirus clade, closely related to Escherichia phage wV8 and Salmonella phage Felix O1, while displaying distinct tail fiber gene signatures associated with host specificity. These findings support the potential of P293 as a candidate for phage-based biocontrol strategies against APEC in poultry production.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00705-026-06602-8.

Introduction

Avian pathogenic Escherichia coli (APEC), a member of the extraintestinal pathogenic E. coli (ExPEC) group, specifically infects avian hosts and induces both respiratory and systemic disease [1]. Traditionally, APEC has been viewed as a secondary or opportunistic pathogen that emerges under conditions of intensive farming, inadequate sanitation or other stress factors such [2]as viral respiratory infections or poor husbandry practices [3]. However, it is now clear that APEC can also act as a primary pathogen in immunocompetent avian hosts [4]. Moreover, its tight linkage to commercial poultry production systems renders APEC a significant reservoir of antimicrobial-resistance determinants. While antibiotics have long been the cornerstone for preventing and treating APEC infections, their widespread misuse and overuse have driven the emergence of multidrug-resistant (MDR) strains [5, 6]. These MDR APEC isolates frequently carry plasmids encoding multiple antibiotic resistance genes (ARGs), which facilitate horizontal dissemination across bacterial populations [7]. To mitigate the acceleration of bacterial antimicrobial resistance, the World Health Organization (WHO), the Food and Agriculture Organization of the United Nations (FAO), and the World Organization for Animal Health (OIE) have launched global initiatives to regulate and reduce the misuse of antibiotics [8]. Consequently, effective control of APEC necessitates a multifaceted approach; the pathogen’s documented epidemiological expansion following restrictions on antimicrobial use, together with mounting regulatory pressures, highlights the urgent need for non-antibiotic intervention strategies.

Bacteriophages are viruses capable of lysing bacteria and are ubiquitous in natural environments. Compared with conventional antibiotics, phage-based antibacterial agents exhibit several notable characteristics: they display high host specificity, targeting only the intended bacterial cells without affecting host tissues or the normal microbiota; they exist in vast numbers in the environment, making nature itself a rich reservoir for therapeutic phages [9]; and they possess unique pharmacokinetic properties in vivo, whereby lytic phages self-amplify at the site of infection as they kill their bacterial hosts [10, 11]. In addressing the challenge of MDR bacteria, phages can exert their distinctive bactericidal effects through multiple mechanisms; for example, by encoding enolase-inhibitory proteins that specifically bind key enzymes in carbohydrate metabolism, thereby disrupting ATP synthesis and the supply of cell-wall precursors, ultimately resulting in bacterial lysis and death [12]. Consequently, the isolation and characterization of novel bacteriophages targeting APEC are critically important for expanding phage libraries and improving strategies to prevent and treat APEC infections.

In 2022, the International Committee on Taxonomy of Viruses (ICTV) formally abolished the morphology-based family-level taxa Podoviridae, Siphoviridae and Myoviridae, along with the order Caudovirales, and introduced a unified binomial species nomenclature [13]. Within the newly established class Caudoviricetes, twenty-two new families were defined, of which fourteen have been assigned to four novel orders. Although the term “myovirus” persists as an informal morphological grouping, its taxonomy has shifted from morphology to genome-based phylogenetic classification. Myoviruses have demonstrated both safety and efficacy in the treatment of multidrug-resistant bacterial infections. For example, Yao et al. isolated the strictly lytic phage PEC9, which protects mice against infection by APEC serotypes O1 and O2 [14], and Naghizadeh et al. showed that a phage cocktail could ameliorate organ lesions in quail infected with APEC [15]. Advances in genetic engineering and synthetic biology for phage modification are poised to accelerate the clinical application of myoviruses in antibacterial therapy [16]. In this study, we isolated and characterized a lytic bacteriophage, Ecolivirus Myo-P293 (abbreviated as P293), from a poultry farm that specifically targets APEC. Transmission electron microscopy (TEM) revealed morphological features consistent with the Myoviridae family. Comprehensive characterization was conducted based on its biological properties and whole-genome sequencing. This study provides a valuable reference for the expansion of APEC-targeting phage libraries and the development of phage-based antimicrobial agents.

Materials and methods

Bacterial strains and phage host range determination

The bacterial strains used in this study were provided by the GuoTai (Taizhou) Center of Technology Innovation for Veterinary Biologicals. A total of 17 bacterial strains were tested, including 15 Escherichia coli (E. coli) strains and 2 Salmonella strains.The E.coli group comprising laboratory strains DH5α, BL21, and Top10, along with 12 serotyped field isolates recovered from poultry farms The Salmonella pullorum (S. pullorum) strain SA40 and Salmonella enteritidis (S. enteritidis) strain S429 were obtained from Guangxi Junke Biotech Co., Ltd. (Beihai, China) (Table 1). All strains were cultured in Luria-Bertani (LB) broth at 37 °C under aerobic conditions. For long-term storage, bacterial stocks were preserved in LB broth supplemented with 30% (v/v) glycerol at -80 °C. The host range of phage P293 was assessed using a spot assay [17]. Briefly, 10 µL of phage suspension (~ 1 × 10⁹ PFU/mL) was spotted onto the surface of LB agar plates overlaid with lawns of each bacterial strain. Plates were incubated at 37 °C for 4–6 h, and lytic activity was evaluated based on the formation of clear lysis zones. To further quantify phage infectivity, the efficiency of plating (EOP) was determined by performing plaque assays using all 15 field E. coli strains and 2 Salmonella strains. The EOP was calculated by comparing the plaque-forming units (PFU) on each susceptible strain with those on the original host strain (E. coli DPE 2).

Table 1.

Host range of bacteriophage Ecolivirus Myo-P293 and efficiency of plating (EOP)

No. of host	Bacterial host	Strains	Serotype	Spot test a	Plaque production ability	Total no. of plaque (pfu/ml) / Efficiency of plating (EOP)	Sources b
1	Escherichia coli	DH5α	-	-	-	-	LS
2	Escherichia coli	BL21	-	-	-	-	LS
3	Escherichia coli	Top10	-	-	-	-	LS
4	Escherichia coli	GPE12	O1	-	-	-	LS
5	Escherichia coli	CPE30	O1	-	-	-	LS
6	Escherichia coli	CPE3	O1	-	-	-	LS
7	Escherichia coli	DPE2	O2	++	+	1.34 × 1010	LS
8	Escherichia coli	DPE14	O2	++	+	1.18 × 1010	LS
9	Escherichia coli	CPE5	O2	+	+	2.45 × 109	LS
10	Escherichia coli	CPE11	O78	+	+	1.73 × 109	LS
11	Escherichia coli	GPE7	O78	-	-	-	LS
12	Escherichia coli	GPE19	O78	-	-	-	LS
13	Escherichia coli	CPE22	O145	+	+	8.82 × 108	LS
14	Escherichia coli	CPE8	O145	-	-	-	LS
15	Escherichia coli	DPE4	O145	-	-	-	LS
16	Salmonella pullorum	SA40	-	-	-	-	Gift
17	Salmonella enteritidis	S429	-	-	-	-	Gift

^a ++ = large clear lysis, + = small clear lysis, - = no lysis of plating

^b LS stands for lab stock and Gift stands for received from Guangxi Junke Biotech Co., Ltd

Phage isolation, purification, and amplification

Phage P293 were isolated from sewage samples collected at a duck farm, using E. coli O2 (DPE 2) as the indicator host strain. Phage isolation was performed according to previously described methods [18], based on the plaque assay technique. To obtain clonal phage populations, individual plaques were picked using sterile micropipette tips and subjected to at least five successive rounds of purification. For phage amplification, the purified phages were enriched as follows: a 50 mL culture of the indicator host (10⁷ CFU/mL) in LB broth was infected with purified phage and incubated at 37 °C with shaking at 120 rpm. After complete lysis, the culture was centrifuged at 6,000 × g for 15 min at 4 °C to remove residual bacterial cells and debris. The resulting supernatant was further centrifuged at 15,000 × g for 1 h at 4 °C to pellet the phage particles. The phage pellets were resuspended in SM buffer (100 mM MgSO₄·7 H₂O, 10 mM NaCl, 50 mM Tris-HCl, pH 7.5) and subsequently filtered through 0.22 μm syringe filters to ensure sterility. Phage titers were determined using the double-layer agar method, which also confirmed the presence of lytic phages in the filtrates [2, 19].

Transmission electron microscopy (TEM)

Approximately 5 µL of purified P293 suspension was applied onto glow-discharged, carbon-coated copper grids (300 mesh) and allowed to adsorb for 1 min. Excess liquid was then carefully removed using filter paper. The grids were subsequently stained with 2% (w/v) uranyl acetate for contrast enhancement and air-dried at room temperature. The negatively stained phage particles were examined using a Hitachi transmission electron microscope (Hitachi, Japan) operated at an accelerating voltage of 80 kV.

Phage adsorption assay

To determine the adsorption kinetics of the P293 to its host, an adsorption assay was performed with minor modifications based on previously described protocols [20]. Briefly, the E. coli DPE 2 host strain was cultured in LB broth and infected with phage at a multiplicity of infection (MOI) of 0.1. The mixture was incubated at 37 °C, and aliquots were collected at 0, 5, 10, 15, 20, 25, and 30 min post-infection. The samples were immediately centrifuged at 10,000 × g for 10 min to pellet the bacterial cells. The resulting supernatants were filtered through 0.22 μm syringe filters to remove residual cells, and the titers of non-adsorbed phages were determined using the double-layer agar method. All experiments were performed in triplicate to ensure reproducibility.

One-step growth curve

The one-step growth experiment was performed with slight modifications based on previously described methods [21]. Briefly, exponentially growing E. coli DPE2 cells were adjusted to 1 × 10⁷ CFU/mL and infected with filtrated P293 lysate at an MOI of 0.01 (final phage concentration 1 × 10⁵ PFU/mL). The 1 mL adsorption mixture in prewarmed LB broth was incubated at 37℃ for 10 min. The mixture was then centrifuged at 10,000 × g for 30 s to remove unbound phages, and the resulting pellet was resuspended in 10 mL of LB broth. The culture was incubated at 37 °C with shaking at 200 rpm. Aliquots of 100 µL were collected every 30 min over a 3-hour period. Each sample was centrifuged at 10,000 × g for 5 minutes to remove bacterial debris, and the phage titers in the supernatants were determined using the double-layer agar method. All experiments were conducted in triplicate. The burst size was calculated as the ratio of the final number of released phage particles to the initial number of infected host cells.

Stability assay

The stability of phage P293 under various physicochemical conditions was evaluated in terms of pH, temperature, and disinfectant exposure [22]. For pH stability, the phage suspension was adjusted to a final concentration of 1 × 10⁹ PFU/mL using buffers with pH values ranging from 3 to 12. Samples were incubated at room temperature for 1, 2, and 3 h. After incubation, residual phage titers were determined using the double-layer agar method. For thermal stability, the phage at a concentration of 1 × 10⁹ PFU/mL was incubated at 20 °C, 40 °C, 60 °C, and 80 °C for 20, 40, 60, and 80 min. Samples were then immediately cooled on ice, and phage titers were measured using the double-layer agar method. For disinfectant stability, the phage was diluted to 1 × 10⁹ PFU/mL in ethanol solutions at final concentrations of 30% and 60% (v/v). The mixtures were incubated at room temperature for 20, 40, 60, and 80 min, followed by titer determination as above. All treatments were conducted in triplicate. Phage viability under each condition was assessed by plaque formation efficiency on the host lawn, with results expressed as viable phage counts relative to the untreated control.

Biofilm clearance assay

To evaluate phage-mediated biofilm clearance [23], E. coli DPE 2 was adjusted to OD₆₀₀ = 1.0 and inoculated into 96-well plates containing 200 µL LB per well (1:1000 dilution). Plates were incubated statically at 37 °C for 24 h to allow biofilm formation. After removing planktonic cells, 200 µL of phage suspension (1 × 10⁸ PFU/mL) in SM buffer (100 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10 mM MgSO₄) was added to each well and incubated for 6, 12, or 24 h. Wells treated with SM buffer alone served as controls. After incubation, wells were washed with PBS, stained with crystal violet, and OD₅₉₅ values were measured as described above. Biofilm clearance was calculated as: Biofilm clearance (%) = [1 − (OD₅₉₅ of phage-treated well / OD₅₉₅ of control well)] × 100.

Biofilm formation inhibition assay

The inhibitory effect of phage P293 on biofilm formation was evaluated using a crystal violet microtiter-plate method with minor modifications from a previous report [24]. Briefly, overnight cultures of E. coli DPE2 were diluted to approximately 1 × 10⁷ CFU/mL in fresh LB broth. Equal volumes of bacterial suspension and phage P293 dilutions were added to 96-well flat-bottom plates to achieve final MOIs of 0.01, 0.1, 1, and 10. Bacteria without phage (MOI = 0) served as the control. After static incubation at 37℃ for 24 h, wells were gently washed twice with PBS to remove non-adherent cells, stained with 0.1% crystal violet for 15 min, rinsed, and air-dried. The bound dye was solubilized with 30% acetic acid, and absorbance was measured at 595 nm. Biofilm formation inhibition was calculated as: Biofilm inhibition (%) = (OD₅₉₅ of control well − OD₅₉₅ of phage-treated well) / OD₅₉₅ of control well×100.

Phage DNA sequencing and bioinformatic analysis

Genomic DNA of phage P293 was extracted using the phenol/chloroform method, as previously described [25]. The purified DNA was diluted to a final concentration of 0.2 ng/µL for whole-genome sequencing. Whole-genome shotgun (WGS) sequencing was conducted to characterize the phage genome. Genomic libraries with varying insert sizes were constructed and subjected to paired-end (PE) sequencing on the Illumina NovaSeq platform. Raw sequencing reads were de novo assembled using SPAdes, and contigs were filtered based on sequencing depth. High-coverage contigs were aligned against the National Center of Biotechnology Information (NCBI) nucleotide (NT) database using BLASTn to identify viral genome sequences [26]. Final genome assembly was refined by base-level error correction using Pilon. Genomic similarity between Ecolivirus Myo-P293 and previously reported phages was assessed using fastANI v1.34 [27]. Gene prediction, functional annotation, and circular genome visualization were performed using Pharokka v1.7.1 [28]. Whole-genome alignments were carried out with MAFFT v7.520 [29], and amino acid sequence alignments of the major capsid and tail fiber proteins were performed using MUSCLE v5.1 [30]. Phylogenetic relationships were inferred using IQ-TREE v2.2.5 based on the aligned sequences [31]. For comparative genomic analysis, genome collinearity and structural conservation among phage P293, Escherichia phage wV8 (GenBank: EU877232.1), and Salmonella phage Felix O1 (GenBank: AF320576.1) were evaluated using Mauve v2.4.0 [32].

Statistical analysis

Data were expressed as mean ± standard deviation (SD) and analyzed using Graph Pad Prism version 9 software. The level of significance was set at (p ≤ 0.05).

Results

Isolation and morphology of phage P293

Using E. coli DPE 2 as the host, a novel lytic bacteriophage, designated Ecolivirus Myo-P293 (hereafter referred to as P293), was isolated from duck farm sewage in Jiangsu, China. Phage P293 produced clear, uniformly sized plaques with an average diameter of approximately 2 mm on a lawn of E. coli DPE 2 (Fig. 1A). The phage was subsequently propagated and purified for morphological examination. TEM revealed that P293 exhibits typical Myoviridae morphology, characterized by an icosahedral head (~ 70 nm in diameter) and a contractile tail (~ 100 nm in length) (Fig. 1B). The presence of a distinct tail sheath and baseplate structure is consistent with features of the Myoviridae family.

Fig. 1.

Morphological characterization of phage Ecolivirus Myo‑P293 (P293). (A) Plaque morphology of phage P293 formed on Escherichia coli DPE 2 lawns after 6 h of incubation at 37 °C, showing clear and round plaques approximately 2 mm in diameter. Scale bar: 5 mm. (B) Transmission electron micrograph (TEM) of negatively stained phage P293, displaying a typical Myoviridae morphology with an icosahedral head (~ 70 nm in diameter) and a contractile tail (~ 100 nm in length). The image was captured at 80 kV with uranyl acetate staining. Scale bar: 100 nm

Latent times and phage burst sizes

Phage adsorption kinetics demonstrated that over 70% of phage particles were adsorbed onto host cells within the first 5 min of incubation. After 30 min, 17.5% of the phages remained unadsorbed in the supernatant (Fig. 2A). To evaluate lytic activity, P293 was added to logarithmic-phase E. coli DPE 2 cultures. The OD₆₀₀ decreased from 0.65 to 0.234 within 3 h and further dropped to 0.189 by 10 h post-infection, accompanied by visible clearing of the culture and lysed bacterial debris, confirming the lytic nature of the phage (Fig. 2B). A one-step growth assay was conducted at 37 °C with an MOI of 0.01 to assess the replication dynamics of P293. The results showed a latent period of approximately 30 min, followed by a large burst size of 244 ± 36 PFU per infected cell (Fig. 2C).

Fig. 2.

Biological characterization of phage Ecolivirus Myo‑P293 (P293). (A) Adsorption kinetics of phage P293 to E. coli DPE 2 at 37 °C. Over 70% of phages adsorbed to host cells within 5 min; unadsorbed phage titers in the supernatant were measured at indicated time points. (B) In vitro bacteriolytic activity of phage P293 against E. coli DPE 2 at an MOI of 1. Bacterial growth was monitored by OD₆₀₀ over 10 h. Phage infection led to a rapid decline in optical density, indicating efficient bacterial lysis. (C) One-step growth curve of phage P293 at an MOI of 0.01. The latent period was approximately 30 min, followed by a sharp increase in phage titer, with a calculated burst size of 244 ± 36 PFU per infected cell. Data are presented as means ± SD from three independent experiments

Host range and lytic activity of phage P293

To evaluate the biological control potential of phage P293, its lytic activity against E. coli strains was assessed using the spot assay (Table 1). A total of 15 E. coli strains were tested, including 12 field isolates representing serotypes O1, O2, O78, and O145, and three laboratory strains (DH5α, BL21, and Top10). Phage P293 exhibited no lytic activity against the three laboratory strains or the O1 serotype isolates. In contrast, it demonstrated clear lytic activity against all three O2 serotype strains, as well as one isolate each from the O78 and O145 serotypes. The number of plaques formed on susceptible strains varied among isolates, indicating strain-specific differences in susceptibility. The host range of P293 was further explored using two representative Salmonella serovars prevalent in poultry, S. pullorum and S. enteritidis. Neither strain was susceptible to lysis by P293, confirming that its host specificity was restricted to APEC.

Stability of phage P293 under different environmental conditions

The environmental stability and persistence of phages are critical factors for their potential application in biological control. To assess the stability of phage P293, its tolerance to varying pH levels, temperatures, and exposure to common disinfectants was evaluated. Phage P293 (1 × 10⁹ PFU/mL) was incubated at pH values ranging from 3 to 12 for 1, 2, and 3 h. As shown in Fig. 3A, the highest phage titer was observed at pH 7, indicating it to be the optimal condition for P293 stability. The phage remained stable under near-neutral and mildly acidic or alkaline conditions, but exhibited significant titer loss under extreme pH environments. Thermal stability assays revealed that P293 was stable at 20 °C to 40 °C, with minimal loss of infectivity. However, phage survival rate decreased significantly at temperatures above 60 °C. As shown in Fig. 3B, treatment at 60–80 °C for 20 min reduced the survival rate from 65% to 24%. Prolonged exposure further compromised phage viability, and complete inactivation was observed after 80 min at 80 °C, indicating that P293 is heat-sensitive but remains stable at moderate temperatures. To assess its sensitivity to disinfectants, P293 was treated with 30% and 60% ethanol for 20, 40, 60, and 80 min (Fig. 3C). At 30% ethanol, phage survival rate dropped to 18% after 60 min. In contrast, 60% ethanol led to near-complete inactivation within the same timeframe. These results suggest that P293 is highly sensitive to ethanol treatment, particularly at higher concentrations and extended exposure durations.

Fig. 3.

Environmental stability of phage Ecolivirus Myo‑P293 (P293). (A) pH stability assay. P293 (1 × 10⁹ PFU/mL) was incubated at various pH values (3–12) for 1, 2, and 3 h at room temperature. The phage titer remained stable between pH 5–9, with maximal activity at pH 7, and declined significantly outside this range. (B) Thermal stability assay. P293 was treated at different temperatures (20–80 °C) for 20, 40, 60, and 80 min. Phage infectivity was stable at 20–40 °C but decreased rapidly above 60 °C, with complete inactivation at 80 °C after 80 min. (C) Ethanol sensitivity assay. P293 was exposed to 30% and 60% ethanol for 20–80 min. Phage viability decreased significantly with increasing ethanol concentration and exposure time, with near-complete inactivation at 60% ethanol for 60 min. All data are presented as means ± SD from three independent experiments

Anti-biofilm effects of phage P293 against E. coli DPE 2

The ability of phage P293 to degrade mature biofilms formed by E. coli DPE 2 was evaluated using a phage suspension at a concentration of 1 × 10⁸ PFU/mL. As shown in Fig. 4A, phage treatment resulted in a significant, time-dependent reduction in biofilm biomass compared with the untreated control. After 24 h of exposure, phage P293 exhibited a pronounced biofilm clearance effect, with a removal efficiency of approximately 45%, indicating its potential to disrupt established E. coli biofilms. To further evaluate the preventive potential of phage P293 against APEC biofilms, a biofilm formation inhibition assay was conducted. When phage P293 was added at the beginning of incubation, biofilm biomass was significantly reduced in a dose-dependent manner (Fig. 4B). The inhibition rates reached approximately 39.4 ± 4.5%, 60.56 ± 1.8%, 79.9 ± 3.4%, and 80.06 ± 1.9% at MOIs of 0.01, 0.1, 1, and 10, respectively, compared with the phage-free control (MOI = 0). These findings demonstrate that P293 not only disrupts pre-formed biofilms but also effectively prevents biofilm establishment during early bacterial growth.

Fig. 4.

Biofilm clearance and inhibition ability of phage Ecolivirus Myo‑P293 (P293). (A) Biofilm clearance assay. Preformed E. coli DPE 2 biofilms were treated with P293 (1 × 10⁸ PFU/mL) for 6, 12, and 24 h. Residual biofilm biomass was quantified using crystal violet staining. P293 treatment led to a time-dependent reduction in biofilm mass, with a clearance rate of approximately 45% after 24 h. (B) Inhibition of biofilm formation by phage P293 added at the beginning of incubation at different MOIs (0.01, 0.1, 1, and 10). Biofilm biomass was quantified by crystal violet staining after 24 h static incubation at 37℃. All data represent the mean ± SD of three independent experiments. Statistical differences compared to untreated controls were determined using Student’s t-test (p < 0.05)

Genomic features and functional annotation of phage P293

The complete genome of phage P293 was determined to be a double-stranded DNA molecule comprising 89,469 base pairs (Fig. 5) (Supplementary File S1). The overall GC content was approximately 38.5%, with a distinct pattern of GC skew observed across the genome. A total of 95 open reading frames (ORFs) were predicted, of which 52 were functionally annotated based on sequence similarity to known phage proteins. Comprehensive genomic annotation data for phage P293, including ORF positions, predicted functions, and PHROG-based classifications, were summarized in Supplementary Table S1. These ORFs were classified into several functional modules, including structural proteins, lysis-related proteins, DNA metabolism and replication, and host interaction functions. Structural and morphogenesis-related genes were primarily clustered in one region and included genes encoding major head protein, tail sheath, tail fiber, baseplate wedge subunits, and portal protein, consistent with the morphological characteristics of Myoviridae phages. Notably, genes such as tail length tape measure protein and neck protein were also identified, suggesting conserved virion assembly architecture. The lysis cassette comprised genes encoding a putative endolysin, RIIA and RIIB lysis inhibitors, and an Rz-like spanin, which are typically involved in host cell lysis through coordinated degradation of the cell wall and outer membrane. Additionally, several ORFs were associated with DNA replication and nucleotide metabolism, including predicted DNA helicases, polymerase subunits, and nucleotide-modifying enzymes. Approximately 40% of the predicted ORFs were annotated as hypothetical proteins with unknown function, suggesting potential novel phage-specific genes requiring further investigation. Comparative analysis revealed high genomic synteny and homology between P293 and members of the Phage01D_k149 group, particularly in regions encoding structural components and lysis-related functions.

Fig. 5.

Circular genome map of phage Ecolivirus Myo‑P293. The complete genome of Ecolivirus Myo‑P293 is 89,518 bp of double-stranded DNA with a GC content of 38.5%. A total of 95 open reading frames (ORFs) were predicted and are displayed on the circular map. Arrows indicate the direction of transcription. ORFs are color-coded by predicted function: structural proteins (blue), DNA replication and regulation (green), host lysis (red), and hypothetical proteins (grey). Functional annotation was performed using Pharokka v1.7.1, and the map was generated based on genomic coordinates and annotation results. No antibiotic resistance or virulence genes were detected. The modular organization is consistent with that of Myoviridae phages, indicating a strictly lytic lifestyle

Comparative genomic analysis of phage P293

To investigate the genomic relatedness of Ecolivirus Myo-P293 with other well-characterized myoviruses, comparative genomic analysis was performed using Mauve alignment software. The genomes of Escherichia phage wV8 and Salmonella phage Felix O1 were selected for comparison due to their phylogenetic proximity and similar genome sizes. As shown in Fig. 6, P293 shares extensive collinear regions with Escherichia phage wV8, particularly across the structural and lysis gene clusters. Large locally collinear blocks (LCBs) indicate a high degree of sequence conservation and genome organization, suggesting that P293 and wV8 may belong to the same genus or closely related lineages. Most conserved regions span from ~ 10 kb to ~ 85 kb, and gene order remains largely syntenic. In contrast, the alignment between P293 and Salmonella phage Felix O1 revealed fewer and less extensive homologous regions. While some conserved sequences were observed in the structural modules, several genomic rearrangements and inversions were evident, especially at the terminal regions. This suggests that although P293 and Felix O1 share some common ancestry, they have diverged considerably in genome architecture. Together, these results indicate that phage P293 exhibits a high degree of genomic similarity and collinearity with E. coli phage wV8, whereas it shares limited homology with Salmonella phage Felix O1, supporting its classification as a novel Escherichia-infecting myovirus with distinct evolutionary lineage.

Fig. 6.

Comparative genomic analysis of phage Ecolivirus Myo‑P293 with related phages. Progressive Mauve alignment of the genomes of Ecolivirus Myo‑P293, Escherichia phage wV8 (GenBank: EU877232.1), and Salmonella phage Felix O1 (GenBank: AF320576.1). Colored blocks represent locally collinear blocks (LCBs), indicating conserved genomic regions among the three phages. The height of each block reflects the degree of nucleotide sequence similarity. Despite overall synteny, notable variation is observed in regions encoding tail fiber and hypothetical proteins, suggesting possible divergence in host specificity and adaptation. The alignment reveals that Ecolivirus Myo‑P293, shares higher genome collinearity with E. coli phage wV8 than with Salmonella phage Felix O1

Phylogenetic analysis of phage P293

To determine the phylogenetic position of phage Ecolivirus Myo-P293, phylogenetic trees were constructed based on its complete genome sequence (Fig. 7A), major capsid protein (Fig. 7B), and tail fiber protein (Fig. 7C). As shown in the genome-based phylogenetic tree (Fig. 7A), P293 clustered within the unclassified Felixounavirus clade, and was most closely related to Escherichia phage EF202P1 and Salmonella phage Felix O1, suggesting a shared evolutionary lineage with this group of lytic Myoviridae phages. Analyses based on the amino acid sequences of the major capsid and tail fiber proteins reflect functional relatedness rather than strict evolutionary ancestry. The major capsid protein tree (Fig. 7B) supported the genomic classification, with P293 grouping closely with Felixounaviruses such as Salmonella phage Felix O1 and Escherichia phage P7, while remaining distinct from members of the T4-like and P2-like virus clades. In contrast, the tail fiber protein tree (Fig. 7C) revealed a slightly divergent pattern: P293 formed a branch near the Felixounavirus cluster but displayed increased sequence distance from closely related Escherichia phages, reflecting the high variability and modular evolution typically observed in tail fiber genes. Collectively, these results suggest that P293 is a member of the unclassified Felixounavirus group, exhibiting close genomic and structural similarities with known Escherichia and Salmonella phages, while maintaining distinct features in its tail fiber region that may contribute to its host specificity and adaptive potential.

Fig. 7.

Phylogenetic analysis of phage Ecolivirus Myo‑P293. (A) Whole-genome-based phylogenetic tree constructed using IQ-TREE v2.2.5, based on alignment with MAFFT v7.520. (B) Phylogenetic tree based on capsid protein amino acid sequences. (C) Phylogenetic tree based on tail fiber protein sequences. All trees were generated using the maximum likelihood method with 1,000 bootstrap replicates. Ecolivirus Myo‑P293 clusters closely with Escherichia phage wV8 (GenBank: EU877232.1) and Salmonella phage Felix O1 (GenBank: AF320576.1), forming a distinct branch within the unclassified Felixounavirus group. Analyses of capsid and tail fiber proteins reflect functional relatedness among structural proteins, while variability in the tail fiber sequence highlights functional divergence in host recognition. Bootstrap values above 70% are shown at the nodes. Scale bars indicate substitutions per site

Discussion

In this study, we report the isolation, characterization, and genomic analysis of P293, a novel Myoviridae phage with potential as a biocontrol agent against APEC. Our results demonstrate that P293 exhibits high lytic efficiency, efficacy against biofilms, and favorable stability-features that align with the functional profile desired for phage-based interventions in poultry.

Phage P293 exhibited strong lytic activity targeting O2 serotypes and select O78/O145 APEC strains, reflecting a relatively narrow yet clinically relevant host range. This specificity is consistent with other APEC-targeting phages, such as vB_EcoP_PW8 [33] and phage AG-MK-2022-Basu [34], which both effectively lyse multidrug-resistant (MDR) strains despite having a restricted host breadth. Notably, P293 achieved a rapid adsorption rate of over 70% within 5 min and released a burst size of approximately 244 PFU per infected cell, which is comparable to or even surpasses other APEC-targeting phages in terms of replicative fitness.

Furthermore, treatment with P293 led to the removal of around 45% of mature E. coli biofilm biomass after 24 h, a result consistent with previous phage-based biofilm disruption studies. For instance, UPWr_E phage cocktails reduced biofilms by 50–80% across surface types [35], and vB_EcoP_PW8 similarly inhibited biofilm formation and maintenance [33]. In addition to disrupting established biofilms, P293 also exhibited a marked inhibitory effect on biofilm formation, showing a progressive increase in inhibition with rising MOI values. These dual activity results support the potential of P293 for applications in farm sanitization and the protection of poultry flocks, especially considering the resistance of APEC biofilms to traditional disinfectants.

The high-quality genomic sequencing revealed a ~ 89.5 kb dsDNA genome encoding 95 ORFs, which are organized into modules associated with the phage’s head, tail, lysis, and replication. About 40% of these ORFs correspond to hypothetical proteins, which are common in other myoviruses with limited functional characterization and warrant further investigation. Comparative genomic analysis with Escherichia phage wV8 and Salmonella phage Felix O1 showed strong synteny in structural and lysis genes, suggesting that P293 shares an evolutionary lineage within the unclassified Felixounavirus clade.

Phylogenetic analysis further highlighted that tail fiber proteins of P293 functionally diverge from closely related Felixounavirus phages, indicating possible adaptive evolution in receptor binding domains. As Tail fibers are critical for host specificity due to their interaction with bacterial surface receptors, such as LPS and O-antigen complexes [36], the variability observed in P293’s tail fiber sequences is consistent with its narrow host range. Engineering tail fiber swaps has been shown to significantly expand host range in related myovirus systems, such as T5-like phages [37], offering a potential strategy to broaden P293’s applicability.

Phage P293 remained stable across a physiologically relevant pH range (5–9) and temperatures (20–40 °C), but was rapidly inactivated at temperatures ≥ 60 °C or in the presence of 60% ethanol, which consistent with findings in phages vB_EcoP_PW8 and AG-MK-2022-Basu [33, 34]. These finding suggest that while P293 is suitable for applications in feed water and sanitation, however, stabilizers may be needed when exposed to heat or ethanol.

For the practical application of P293 in poultry biocontrol, several avenues warrant further exploration. In vivo challenge trials are critical to assess its therapeutic potential. Previous work with phage AG-MK-2022-Basu have demonstrated protection against colibacillosis in broilers, showing improved body weight gain and reduced bacterial load [38]. A similar approach with P293 could help confirm its therapeutic potential. Additionally, engineering the tail fiber gene using directed evolution or recombination strategies could widen host range without compromising infection efficiency [39]. Lastly, the development of phage cocktails that combine P293 with other phages may mitigate resistance development and broaden its therapeutic scope [40].

Conclusions

This study presents the isolation and characterization of the novel lytic phage, Ecolivirus Myo‑P293, targeting avian pathogenic Escherichia coli. Phage P293 exhibits rapid adsorption, strong lytic activity, significant biofilm clearance, and desirable thermal and pH stability under physiological conditions. Genomic and phylogenetic analyses indicate that P293 belongs to the Myoviridae family and closely related to Felixounavirus phages. These promising attributes suggest that P293 is a potential candidate for the development of alternative antibacterial agents aimed at combating APEC infections in poultry.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Hai Xu and Yaming Feng conceived the project, Qiurong Qi and Yalu Zhu conducted the majority of the experiments and analyses. Erzhong Wu and Guiqin Yan were responsible for the collection of clinical samples. Hai Xu and Yu Lu drafted the manuscript, and all authors reviewed and approved the final version of the manuscript.

Funding

This work was supported by the Jiangsu Agricultural Science and Technology Independent Innovation Fund (CX (24) 2005).

Data availability

The raw sequencing data for Ecolivirus Myo‑P293 have been deposited in the NCBI SRA under BioProject accession number PRJNA1302722.

Conflict of interest

We declare no conflict of interest.