This study used 26 bacterial strains, including 22 E. coli strains and 4 Salmonella strains, all isolated and identified by the Laboratory of Pathogenic Mechanisms and Control of Subtropical Livestock Diseases at Guangxi University or obtained through procurement (Table 1). The original bacterial strains were preserved at −80 °C. All bacteria were cultured in Luria-Bertani (LB) broth supplemented with 0.9% (w/v) NaCl at 37 °C.

The two bacteriophages, vB_EcoM_BYEP01 (infecting E. coli BYEC01) and vB_SalS_SP14 (infecting S. enterica ATCC 14028), were independently isolated from a single swine wastewater sample collected from a local farm. The water sample was centrifuged at 8,000×g for 10 min and then filtered through a 0.22 μm membrane filter to remove bacterial cells. The filtrate was enriched by mixing it with an early log-phase culture of the corresponding host bacterium and double-strength LB broth, then incubating at 37 °C with shaking for 12–18 h. After enrichment, the lysate was centrifuged and filtered. The presence of phages was confirmed using the spot test. Individual, well-isolated plaques were selected and purified at least thrice to ensure clonal purity.

Plaque morphology was assessed on double-layer agar plates after 18 h of incubation at 37 °C. The size, transparency, and presence of halos in at least 20 plaques were meticulously recorded. For transmission electron microscopy (TEM), purified phage suspensions were negatively stained with 2% (w/v) uranyl acetate. The grids were examined using a Hitachi HT-7700 TEM at 80 kV. Morphological classification was performed according to the guidelines established by the International Committee on Taxonomy of Viruses.

The lytic spectrum of each phage was assessed against a panel of bacterial strains using a spot test. Briefly, 5 μL of a high-titer phage lysate (≥ 10⁸ PFU/mL) was applied to a lawn of the target bacteria. After incubation, the plates were examined for plaque formation and lysis.

The optimal MOI was determined by infecting mid-log-phase host cultures at various MOIs (10⁻³, 10⁻², 10⁻¹, 10⁰, 10¹, 10², and 10³). The phage-bacterial mixtures were incubated at 37 °C for 20 min to facilitate adsorption. The mixtures were then centrifuged to remove unadsorbed phages. The resulting pellets were re-suspended in fresh LB broth and incubated for 4–5 h. Following incubation, the lysates were centrifuged, and the supernatants were titrated. The MOI that produced the highest phage titer was considered optimal.

The incubation period and outbreak size were characterized using a one-step growth experiment. Briefly, when the MOI was set to 0.1, the phage-bacterial mixture was allowed to adsorb for 10 min. Subsequently, the mixture was centrifuged, re-suspended in fresh pre-heated broth, and incubated at 37 °C. Samples were collected every 10 min (at 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 min) for immediate titration. The incubation period was determined from the growth curve, and the outbreak size was quantified as the ratio of the final phage titer after the ascending phase to the initial number of infected bacteria.

The thermal stability of the phages was evaluated by incubating 1 mL of phage suspension (approximately 10⁸ PFU/mL) in a water bath at 30, 40, 50, 60, 70, 80, and 90 °C. Aliquots were collected at 20, 40, and 60 min intervals, immediately cooled on ice, and subsequently titrated. The phage titer at each time point was expressed as a percentage of the initial value.

The pH stability of the phages was assessed by incubating 1 mL of phage suspension (approximately 10⁸ PFU/mL) in a water bath at pH values ranging from 1 to 14. Aliquots were collected at 20-, 40-, and 60-min intervals and titrated. The phage titer at each time point was expressed as a percentage of the initial titer.

Genomic DNA was extracted from purified high-titer phage lysates using a Phage DNA Isolation kit. Sequencing was performed using the Illumina NovaSeq 6,000 platform with a 150-bp paired-end approach. Raw reads were quality-trimmed and assembled into contiguous sequences using SPAdes software (version 3.15.5). Open reading frames were predicted using Prokka (version 2.6.1) or RAST (Brettin et al., 2015), and functional annotation was performed by searching the NCBI Non-redundant, Swiss-Prot, and conserved domain databases. tRNA genes were identified using tRNAscan-SE (version 2.0.9). Complete genome sequences were deposited in GenBank under accession numbers.

The conserved gene sequences of bacteriophages were selected for functional analysis using NCBI BLAST. The 20 most homologous bacteriophage sequences were identified and analyzed using MEGA7 software. Multiple sequence alignment was performed using Muscle, and the phylogenetic tree was constructed using the neighbor-joining method.

The host strains, E. coli BYEC01 and S. enterica ATCC 14028, were cultured in Mueller-Hinton Broth at 37 °C with agitation at 180 rpm to test the phage-antibiotic synergistic effect. In this study, antibiotics were selected based on their sensitivity or resistance profiles: Polymyxin B (sensitive to E. coli BYEC01), Ampicillin (resistant to E. coli BYEC01 but sensitive to S. enterica ATCC 14028), and Sulfafurazole (resistant to S. enterica ATCC 14028). The preparation and inoculation of the culture medium were performed according to the K-B method, and the operational requirements were outlined in the EUCAST European Union drug-sensitivity testing standards.

All food samples were confirmed to be free of the target pathogens before artificial contamination. The food samples were subsequently contaminated with suspensions of the target pathogens, resulting in final concentrations of approximately 10⁵ CFU/mL in both milk and pig skin. The contaminated samples were air-dried in a biosafety cabinet for 30 min to facilitate bacterial adherence to the surface. Subsequently, the samples were treated with a phage suspension in SM buffer to achieve final phage concentrations of 10⁶ and 10⁷ CFU/mL. The control group received an equivalent volume of sterile SM buffer. The processed milk and pig skin samples were stored at 4 °C and 25 °C, simulating common storage conditions. Samples were collected at predetermined time points (1, 3, 6, and 12 h) for bacterial enumeration. The high MOIs (1,000 and 10,000) were utilized in this initial proof-of-concept study to rigorously evaluate and demonstrate the maximum bactericidal potential of the phages under exaggerated conditions, rather than to reflect typical levels of natural contamination.

Two-week-old Kunming mice, free of specific pathogens, were randomly divided into three groups (10 mice per group): (1) phage treatment, (2) infection control, and (3) simulated infection control (phosphate-buffered saline (PBS) treatment). The target pathogens, either 2 × 10⁸ CFU/mL of E. coli BYEC01 or S. enterica ATCC 14028, were, respectively, gavaged into the phage treatment and infection control groups. Two hours post-infection, the mice in the phage treatment group were administered 400 μL of phage solution at a concentration of 2 × 10⁸ CFU/mL via gavage, whereas those in the infection control group were administered an equivalent volume of sterile PBS.

The survival rate and weight changes of all the mice were recorded daily at consistent times. After 48 h of intragastric inoculation, five mice from each group were euthanized by cervical dislocation. Spleen and intestinal tissues were collected aseptically for further analysis.

Twenty healthy Kunming mice, aged 2–3 weeks, were selected and randomly assigned to four groups (n = 5 per group). Mice in the experimental groups received an intraperitoneal injection of 0.2 mL of phage solution at a titer of 10⁸ PFU/mL, whereas the control group received an equal volume of sterile phosphate-buffered saline (PBS). All animals were maintained under identical environmental conditions. Seven days post-injection, the mice were euthanized by cervical dislocation, and organs were harvested for histopathological examination to assess potential pathological alterations.

The experimental data were statistically processed and expressed as mean ± standard deviation (SD). The difference between the experimental and control groups was determined using Student’s t-test, and the difference between the two experimental groups was determined using Tukey’s test for multiple comparisons using the Statistical Package for the Social Sciences software (version 17.0). Statistical significance was set at p < 0.05.

The two bacteriophages, vB_EcoM_BYEP01 and vB_SalS_SP14, were successfully isolated from a single swine wastewater sample using E. coli BYEP01 and S. enterica ATCC 14028 as host strains. Initial plating on LB agar revealed distinct plaque morphologies for all phages. Bacteriophage vB_EcoM_BYEP01 formed small (0.4 mm in diameter) and clear plaques, implying its potential ability to lyse bacteria (Figure 1A). Conversely, plaques produced by phage vB_SalS_SP14 were larger (2 mm) and clear, surrounded by a sharp halo (Figure 1B).

TEM was used to elucidate their structural differences further. Based on plaque morphology, TEM imaging confirmed that the two bacteriophages belonged to different families. Bacteriophage vB_EcoM_BYEP01 features an icosahedral head and a long, contractile tail, classifying it as a member of the Myoviridae family (Figure 1C). Meanwhile, vB_SalS_SP14 was characterized as an icosahedral structure devoid of a tail and exhibited features typical of the Microviridae family (Figure 1D). This morphological divergence was the first indication of distinct evolutionary lineages.

Subsequently, the lytic capabilities of the bacteriophages were quantitatively assessed by determining their one-step growth parameters and optimal MOI. The one-step growth curve revealed that bacteriophage vB_EcoM_BYEP01 exhibited a latent period of 10 min and a burst size of approximately 70 plaque-forming units (PFU) per infected cell (Figure 1E). Bacteriophage vB_SalS_SP14 exhibited a slightly longer latent period of 20 min and a comparable burst size of approximately 23 PFU/infected cell (Figure 1F). The optimal MOI was found to be 1 for both phages, yielding the highest progeny phage titer (Figure 1G). Notably, the maximum phage yield was achieved at an MOI of 1 for both phages, this observation aligns with the classical principle that an MOI favoring predominantly single infections minimizes inter-viral competition for host cellular resources and avoids premature lysis, thereby maximizing burst size and overall progeny production. These results indicate that, despite their structural divergence, both phages are efficient lytic agents with robust reproductive potentials.

Finally, the stability of the two bacteriophages was assessed at varying pH and temperature conditions. Both bacteriophages vB_EcoM_BYEP01 (Figure 1H) and vB_SalS_SP14 (Figure 1I) demonstrated robust tolerance to both acidic and basic conditions, exhibiting significant stability within a pH range of 6–10. The impact of temperature on bacteriophage vB_EcoM_BYEP01 is illustrated in Figure 1J. Within the temperature range of 30–60 °C, the phage titer remained relatively stable across different time points (Figure 1J). The impact of temperatures ranging from 30 to 50 °C on the bacteriophage vB_SalS_SP14 was minimal (Figure 1K).

In this study, we analyzed the biological characteristics of two newly isolated bacteriophages and revealed that both are lytic phages. The isolation and identification of novel lytic bacteriophages as alternatives to antibiotics can effectively eliminate bacteria. Furthermore, this study establishes a basis for improving the diversity of bacteriophage libraries and facilitates in-depth investigations into the functions of bacteriophage genes.

To investigate the host range of the bacteriophages, 21 bacterial strains from various sources, along with five standard strains, were collected from regions including Guangxi, Guangdong, and Sichuan in China. The 22 E. coli strains comprised: (i) 21 environmental isolates obtained from the same porcine wastewater source as the phages, representing the local and contemporary bacterial population; and (ii) one reference strain (E. coli CVCC 1527) acquired from a national culture collection. Similarly, the four Salmonella strains were all reference type strains obtained from major international culture collections: CVCC 3384, CVCC 1806, ATCC 14028, and CMCC 50746. This selection intentionally included strains of diverse serotypes and geographic origins, enabling us to assess the efficacy of the phages against clinically relevant Salmonella types that extend beyond their primary host range, using well-characterized isolates. The findings revealed that the bacteriophages vB_EcoM_BYEP01 could lyse E. coli strains BYEC01, BYEC02, BYEC04, BYEC05, BYEC06, and BYEC07. Conversely, bacteriophage vB_SalS_SP14 exhibited the ability to lyse only S. enterica ATCC 14028 (Table 1).

Analysis of the phage lysis spectra demonstrated that the lysis profiles of the two phages isolated in this study were relatively narrow, indicating a high degree of host specificity.

To elucidate the genetic basis underlying the distinct morphologies and shared lytic efficiency of vB_EcoM_BYEP01 and vB_SalS_SP14, we sequenced and analyzed their genomes.

The genome of vB_EcoM_BYEP01 was characterized as a double-stranded DNA molecule of 169,509 bp with a G + C content of 35.27%. Through analysis of the VFDB database, no toxin-related genes were identified in the genome of vB_EcoM_BYEP01 (Figure 2A). Conversely, the vB_SalS_SP14 genome was significantly smaller, consisting of a 5,513 bp single-stranded DNA molecule with a higher G + C content of 44.93%. Through analysis of the VFDB database, no toxin-related genes were identified in the genome of vB_SalS_SP14 (Figure 2B). Furthermore, tRNA scan identified seven tRNA genes within the vB_EcoM_BYEP01 genome, whereas no such genes were identified in the vB_SalS_SP14 genome. Further analysis revealed that no known antibiotic resistance genes were detected in the genomes of vB_EcoM_BYEP01 and vB_SalS_SP14 following comprehensive screening against the CARD and ResFinder databases (Figures 2A,B). These fundamental genomic features underscore the significant differences between the two phages. The bacteriophage vB_EcoM_BYEP01 data were successfully uploaded to the NCBI database with accession number OR757453.1. Furthermore, the bacteriophage vB_SalS_SP14 data were submitted to the NCBI database under the accession number PP501405.1.

Phylogenetic analyses were used to determine their evolutionary relationships. A phylogenetic tree constructed from the conserved major capsid protein placed vB_EcoM_BYEP01 within the Tevenvirinae subfamily of Myoviridae (Figure 2C), whereas vB_SalS_SP14 was within the Bullavirinae subfamily of Microviridae (Figure 2D). Consistent with this, a BLAST comparison of the complete genomes revealed no significant similarities between the two phages, conclusively classifying them into two separate genera that evolved from distinct lineages.

A comparative analysis of the two phage genomes highlights their distinct evolutionary paths. Phage vB_EcoM_BYEP01 harbors a large (~169 kbp), structurally complex genome characteristic of members of the Myoviridae family, with multiple genes organized into well-defined functional modules involved in DNA metabolism, virion assembly, and host cell lysis. In contrast, the genome of vB_SalS_SP14 is compact (~5.5 kbp) and streamlined, a feature commonly observed among Microviridae. Despite its reduced size, essential genes encoding the terminase (gpA), portal protein (gpH), major capsid protein (gpC), and tail components (protein D, gpF) are readily identifiable and exhibit conserved synteny (Figure 2B). This striking genomic divergence underscores the convergent evolution of lytic functionality from fundamentally different genetic architectures.

Before performing experiments to evaluate the inhibitory effects of phages in combination with antibiotics on bacterial populations, we examined the in vitro lytic activities of the bacteriophages vB_EcoM_BYEP01 and vB_SalS_SP14 at various MOI. The bacteriophage vB_EcoM_BYEP01 effectively inhibited bacterial growth across an MOI range of 1–1,000, maintaining bacterial density at baseline levels throughout the experiment (Figure 3A). The lytic spectrum of bacteriophage vB_SalS_SP14 was also influenced by the MOI. Specifically, when the MOI ranged from 100 to 1,000, the phage demonstrated significant efficacy in inhibiting bacterial growth (Figure 3B).

After establishing the potent lytic efficacy of the individual phages in vitro, we investigated their potential for combination therapy with conventional antibiotics, a strategy aimed at improving antibacterial activity and mitigating resistance development. First, the antimicrobial resistance profile of the host bacteria was determined using the EUCAST European Antimicrobial Susceptibility Testing standard to evaluate the efficacy of the antibiotic control group against the target bacterial strain (Figure 3C). Subsequently, we used the antibacterial activity curve to assess the inhibitory efficacy of bacteriophages (vB_EcoM_BYEP01 or vB_SalS_SP14) against sensitive and resistant antibiotics in their respective host bacteria.

The antibacterial effect of bacteriophage vB_EcoM_BYEP01 in combination with Polymyxin B (a sensitive antibiotic) on E. coli BYEC01 is illustrated in Figure 3D. Compared to the control group, Polymyxin B and phage groups demonstrated significant inhibition of bacterial growth, with enhanced efficacy observed when antibiotics and phages were used together. There was minimal variation among the combination groups at different antibiotic concentrations (Figure 3D). The antibacterial effect of bacteriophage vB_EcoM_BYEP01 combined with Ampicillin (a resistant antibiotic) on E. coli BYEC01 is presented in Figure 3E. Compared to the control group, the Ampicillin group did not exhibit a significant bactericidal effect. Although the use of bacteriophage vB_EcoM_BYEP01 alone significantly suppressed bacterial growth, its combination with Ampicillin demonstrated a enhancement effect (Figure 3E). These findings indicate that bacteriophage vB_EcoM_BYEP01 exhibits a certain degree of enhancement activity with Ampicillin; however, its enhancement effect when combined with Polymyxin B was particularly significant.

The antibacterial effect of bacteriophage vB_SalS_SP14 in combination with Ampicillin (sensitive antibiotic) on S. enterica ATCC 14028 is illustrated in Figure 3F. Compared to the control group, Ampicillin and phage groups significantly suppressed bacterial growth, with a more pronounced effect observed when antibiotics and phages were used in conjunction (Figure 3F). The antibacterial effect of bacteriophage vB_SalS_SP14 combined with Sulfafurazole (resistant antibiotic) against S. enterica ATCC 14028 is depicted in Figure 3G. Compared to the control group, Sulfafurazole treatment did not exhibit a significant bactericidal effect. However, the combination of phages and antibiotics effectively inhibited bacterial growth, particularly at a concentration of 10 mg/L Sulfafurazole, which demonstrated superior efficacy (Figure 3G). These findings suggest that bacteriophage vB_SalS_SP14 exhibits a certain degree of enhancement activity with Sulfafurazole, whereas its enhancement effect with Ampicillin is even more pronounced.

These results clearly demonstrate that the bactericidal activity is significantly enhanced when the two bacteriophages are administered in combination with antibiotics at sublethal concentrations. This enhancement effect facilitates a more rapid and complete eradication of the bacterial population.

To evaluate the potential of vB_EcoM_BYEP01 and vB_SalS_SP14 as biological control agents for practical use, we investigated their inhibitory effects on the host bacteria in contaminated food samples. Milk and pig skin were selected as representative models for the liquid and solid food substrates, respectively. The food samples were inoculated with their corresponding host bacteria and treated with two doses of the respective phage 10⁶ PFU/mL (MOI = 1,000) and 10⁷ PFU/mL (MOI = 10,000). The samples were then stored at 4 °C (refrigerated) and 25 °C (room temperature) to simulate common storage conditions.

At 4 °C, phage therapy demonstrated significant and sustained antibacterial activity. In milk, compared to the untreated control group, a dose of 1,000 MOI vB_EcoM_BYEP01 reduced the viable E. coli count by 0.96 orders of magnitude after 12 h, whereas a dose of 10,000 MOI vB_EcoM_BYEP01 resulted in a reduction of 1.3 orders of magnitude after the same duration (Figure 4A). Similarly, at 4 °C in milk, the administration of 1,000 MOI of vB_SalS_SP14 resulted in a decrease in viable Salmonella counts by 0.8 orders of magnitude, whereas at a concentration of 10,000 MOI, vB_SalS_SP14 achieved a reduction of 1.04 orders of magnitude. Bacterial growth was effectively inhibited throughout the entire course of the experiment (Figure 4B). Furthermore, no significant change was observed in the number of live bacteria on pig skin stored at 4 °C within the control group. Conversely, a persistent decrease in viable bacteria was observed in the vB_EcoM_BYEP01 treatment group (Figure 4C). Similarly, treatment with vB_SalS_SP14 on pig skin at this temperature demonstrated significant antibacterial efficacy (Figure 4D).

Lower temperatures seem to decelerate both bacterial metabolism and phage replication. However, phages effectively impede pathogen proliferation during refrigerated storage, highlighting their efficacy as protective agents.

More dynamic and rapid bactericidal effects were observed at 25 °C. In milk, the use of 10,000 MOI vB_EcoM_BYEP01 significantly suppressed E. coli activity within 12 h (Figure 4E). Similarly, treatment with 10,000 MOI vB_SalS_SP14 resulted in a significant reduction in Salmonella activity after 12 h (Figure 4F). For E. coli on pig skin, vB_EcoM_BYEP01 also significantly inhibited bacterial activity (Figure 4G). Similarly, for Salmonella on pig skin, treatment with vB_SalS_SP14 resulted in a significant decrease in Salmonella activity within 12 h, maintaining a significantly lower bacterial load than the rapidly increasing control group (Figure 4H). At this environmental temperature, the enhanced efficacy may be attributed to active phage replication and an accelerated lysis cycle that effectively surpasses the bacterial proliferation.

Collectively, these findings underscore the practical significance of both phages as promising biocontrol agents. They effectively limit the survival and growth of target pathogens across different food types and at clinically relevant storage temperatures, providing a targeted strategy for improving food safety.

Before establishing the in vitro infection model, we assessed the safety of the two phages. Phage solutions were administered intraperitoneally to mice over a period of 7 days. No adverse reactions were observed in any of the animals, and postmortem examinations revealed no tissue or organ lesions. These findings indicate that the two phages exert no toxic effects in mice. The mouse survival rate is presented in Figure 5A.

The therapeutic potential of vB_EcoM_BYEP01 and vB_SalS_SP14 was assessed in vivo using mouse infection models. The lethal doses of each pathogen were administered to the mice via gavage. Two hours post-infection, the corresponding phage (1 × 10⁹ PFU) or PBS was administered by gavage for control purposes.

The phage therapy experiment in mice was meticulously monitored for 7 days. The survival outcomes were unambiguous. The survival rate in the E. coli infection group was 100%, with no fatalities in the mice. Conversely, the Salmonella infection group exhibited a survival rate of 60%, whereas no deaths occurred in the treatment group (Figure 5B). Furthermore, there was no significant decrease in body weight within the phage treatment group, indicating robust treatment tolerance and effective prevention of systemic deterioration in control animals (Figure 5C).

To quantitatively evaluate the bactericidal efficacy of bacteriophages in vivo, we measured the bacterial load in the primary target organs 48 h post-infection. In the control group, a significantly high level of bacterial colonization was observed in the spleens and intestines. Conversely, phage therapy significantly reduced bacterial burden. In the vB_EcoM_BYEP01 treatment group, the E. coli load in both the spleen and intestine decreased by 2.09 and 2.13 orders of magnitude, respectively, compared to the control group (Figure 5D). Similarly, treatment with vB_SalS_SP14 resulted in a reduction of 1.02 orders of magnitude for Salmonella in the spleen and a significant decrease of 3.32 orders of magnitude for Salmonella in the intestine (Figure 5E). These findings indicate that bacterial counts in the phage treatment group approached or fell below the detection limit, thereby confirming the effective clearance of pathogens from the systemic circulation and major organs.

Collectively, these in vivo findings demonstrate that a single dose of either vB_EcoM_BYEP01 or vB_SalS_SP14 is safe and significantly effective, achieving 100% survival by rapidly controlling and clearing lethal bacterial infections in a systemic model.