Characteristics, Whole Genome Analysis of a Virulent Phage from Avian-Derived Enterococcus faecalis and Its Application in Poultry Product Processing Safety

TL;DR

A new virulent phage was isolated from poultry sewage and shown to effectively control E. faecalis contamination in poultry products.

Contribution

The study identifies and characterizes a novel phage with strong lytic activity and biosafety for poultry product safety.

Findings

The phage vB-Efa1 belongs to the Siphoviridae family and has a genome of 166,586 bp with no antibiotic resistance genes.

Phage titer is significantly higher at 25 °C than at 4 °C, indicating temperature's key role in its proliferation.

The phage shows good stability at 25–37 °C, pH 6–8, and 4 °C, with a latent period of 30 min and a titer of 8.87 lg PFU/mL.

Abstract

To explore high-quality phage resources for controlling Enterococcus faecalis (E. faecalis) contamination, a virulent phage vB-Efa1 was isolated and purified from poultry slaughterhouse sewage in this study. Its biological characteristics, whole-genome features, and potential in ensuring poultry product processing safety were systematically investigated. The phage belongs to the Siphoviridae family, with an optimal multiplicity of infection (MOI) of 0.1 and a titer of 8.87 lg PFU/mL; it has a 30 min latent period and stable lytic activity, retaining good stability at 25–37 °C, pH 6–8, and 4 °C. Its circular whole genome is 166,586 bp in length with a GC content of 35.46%, encoding 276 genes; no antibiotic resistance genes were detected, and only one low-pathogenic-risk virulence-related sequence was identified. Application tests in poultry products revealed that temperature is the key factor regulating phage titer: the titer stably maintained 5.5–6.6 lg PFU/mL at 4 °C, while proliferating significantly at 25 °C, reaching 7.55–8.38 lg PFU/mL at 12 h. Collectively, vB-Efa1 exhibits superior biological traits, environmental adaptability, and biosafety, making it a promising biocontrol candidate for mitigating E. faecalis contamination in poultry products.

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Full text

1. Introduction

Enterococcus faecalis, a typical foodborne opportunistic pathogen, is widely distributed in the natural environment, as well as in the intestinal tracts and mucosal surfaces of poultry. It can contaminate poultry meat, eggs, and various poultry products through multiple links such as poultry slaughtering, cutting, and processing [1]. This strain exhibits strong environmental adaptability, being able to tolerate low temperature, high salt, and partial disinfectant treatments during poultry product processing. It can survive and reproduce continuously during refrigeration, transportation, and shelf life, which not only causes spoilage and deterioration of poultry products, reduces product quality and shelf life, but also may induce foodborne diseases such as nausea, vomiting, and diarrhea in consumers. For immunocompromised populations, it is more likely to trigger severe complications such as sepsis and endocarditis, seriously threatening the safety of poultry product consumption and bringing huge economic losses to the poultry breeding and processing industries [2,3].

In recent years, with the extensive use and even abuse of antibiotics in the poultry breeding industry, the detection rate of multidrug-resistant Enterococcus faecalis strains (resistant to vancomycin, ampicillin, tetracycline, etc.) in poultry intestines and the poultry product chain has been increasing year by year. Traditional antibiotic-based prevention and control methods have gradually become ineffective, and antibiotic resistance genes are prone to horizontal transfer to human pathogens through the food chain, further exacerbating food safety risks. Therefore, the development of new, safe, and efficient technologies for preventing and controlling pathogenic bacteria in poultry products has become an urgent demand of the industry [4].

As a type of virus that specifically infects bacteria, phages possess unique advantages such as high specificity, lysing only host pathogenic bacteria without damaging beneficial microflora in poultry products, no chemical residues, easy degradation, and low tendency to induce drug resistance. Thus, they are regarded as ideal biological agents to replace antibiotics for controlling pathogenic bacteria in the field of poultry product processing [5,6]. Among them, virulent phages can quickly invade host bacteria, complete the replication-lysis cycle, and release progeny phages to continuously exert bacteriostatic effects. They can effectively inhibit the contamination and proliferation of Enterococcus faecalis in all links of poultry product processing and storage, showing broad application prospects in poultry product processing safety control, preservation and color retention, and have become a research hotspot in the fields of food microbiology and poultry product processing [7].

At present, although studies on Enterococcus faecalis phages have been reported, most of them focus on clinical or environmental strains. The exploration of specific phage resources targeting avian-derived Enterococcus faecalis is insufficient, and systematic research on their biological characteristics, genetic background, and practical application potential in poultry product processing environments (such as different temperatures, pH values, and processing technologies) is relatively scarce, which greatly limits their industrial application in the prevention and control of pathogenic bacteria in poultry products.

Based on this, a virulent phage vB-Efa1 targeting avian-derived Enterococcus faecalis was isolated and purified from the sewage of a poultry slaughterhouse in this study. Its core biological characteristics, including lytic spectrum, optimal multiplicity of infection (MOI), one-step growth curve, temperature and pH stability, were systematically investigated to clarify its adaptability in poultry product processing and storage environments. Meanwhile, whole-genome sequencing and bioinformatics analysis were performed to resolve its genomic structure, functional gene annotation, and evolutionary characteristics, as well as to screen for safety risk factors such as antibiotic resistance genes and virulence genes. This study aims to explore high-quality phage resources suitable for the safety control of poultry product processing, provide theoretical basis and technical support for the development of new biological bacteriostatic agents for poultry products and the construction of a technical system for preventing and controlling Enterococcus faecalis contamination in poultry products, and help improve the safety level of poultry product processing.

2. Materials and Methods

2.1. Sources of Strain and Phage

The Enterococcus faecalis strain used in this experiment was isolated from chicken intestinal tissue samples of a poultry slaughterhouse, serving as the host bacterium for phage isolation and identification. Phage isolation samples were obtained from the sewage of a poultry slaughterhouse located in Jiangsu Province. After collection, the samples were placed in sterile sampling tubes, transported to the laboratory under 4 °C refrigeration, and used for subsequent phage isolation and screening experiments.

2.2. Main Reagents and Consumables

Main reagents: LB broth medium, LB agar powder (Qingdao Haibo Biotechnology Co., Ltd., Qingdao, China); SM buffer (50 mmol/L Tris-HCl, 10 mmol/L MgSO_4_·7H_2_O, 100 mmol/L NaCl, pH 7.5, Qingdao Changhe Biotechnology Co., Ltd., Qingdao, China); sterile normal saline (0.85% NaCl solution, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China); other chemical reagents were of analytical grade, purchased from regular domestic reagent suppliers. Main consumables: 0.22 μm disposable syringe filters (Merck Millipore, Darmstadt, Germany); conventional experimental consumables such as sterile sampling tubes, centrifuge tubes, and Petri dishes were purchased from Qingdao Jindian Biotechnology Co., Ltd., Qingdao, China.

2.3. Isolation and Purification of Phage

Fifty milliliters of sewage sample was placed in a sterile centrifuge tube, centrifuged at 7000 r/min for 30 min, and 50 mL of supernatant was collected. The supernatant was filtered and sterilized using a 0.22 μm disposable syringe filter to remove bacteria and impurities. Add 50 mL of LB liquid medium and 50 mL of the filtered sewage supernatant into a 200 mL conical flask sequentially, and 300 μL of logarithmic-phase Enterococcus faecalis culture (concentration: 1 × 10^8^ CFU/mL), followed by shaking cultivation at 37 °C and 200 r/min for 24 h in a constant-temperature shaking incubator for phage enrichment. The enriched culture was centrifuged at 5000 r/min for 10 min (BECKMAN COULTER Allegra C-34R), and the supernatant was collected and re-filtered with a 0.22 μm filter to obtain crude phage extract, which was stored at 4 °C for later use [8].

Phage purification was performed by the double-layer agar plate method: The crude phage extract was serially diluted 10-fold with SM buffer to obtain dilutions ranging from 10^1^ to 10^8^. One hundred microliters of each dilution was mixed with 100 μL of logarithmic-phase Enterococcus faecalis culture in a sterile centrifuge tube, gently mixed, and incubated at room temperature for 1 min to allow sufficient adsorption of phage to host bacteria. Subsequently, 4 mL of melted 0.7% LB semi-solid agar cooled to 50 °C was added, quickly inverted and mixed, and immediately poured onto a solidified 1.4% LB solid agar plate. After the semi-solid agar was completely solidified, the plate was inverted and cultured at 37 °C for 12 h in a constant-temperature incubator, and the morphology of plaques was observed and recorded. A single plaque with uniform morphology and clear edges was picked with a sterile 10 μL pipette tip, inoculated into 300 μL of SM buffer, and incubated at room temperature for 3 h to allow full release of phage. The phage release solution was subjected to 5 repeated purification steps using the aforementioned double-layer agar plate method until a single plaque with consistent morphology was obtained, indicating the purified virulent phage, named vB-Efa1, which was stored at 4 °C for later use.

2.4. Observation of Phage Morphology

Under sterile conditions, the purified phage solution was concentrated by ultracentrifugation (100,000 r/min, 4 °C, 2 h). The concentrated phage sample was dropped onto a copper grid, negatively stained with 2% phosphotungstic acid (pH 7.0) for 10 min, air-dried naturally, and sent to Wuhan Sai’erwei Biotechnology Co., Ltd., Wuhan, China for morphology observation using a transmission electron microscope (TEM). Key structural parameters such as head diameter and tail length were measured [8]. Before the experiment, the titer of the phage solution was verified by the double-layer agar plate method to ensure a titer of ≥1 × 10^8^ PFU/mL.

2.5. Determination of Optimal Multiplicity of Infection (MOI)

Logarithmic-phase Enterococcus faecalis culture (concentration: 1 × 10^8^ CFU/mL) was added to sterile centrifuge tubes, and purified phage solution was added according to different MOI values. After vortex mixing for 30 s, the tubes were shake-cultured at 37 °C and 200 r/min for 3.5 h in a constant-temperature shaking incubator. After cultivation, each group of samples was centrifuged at 7000 r/min for 5 min, the supernatant was collected and filtered with a 0.22 μm filter, and the phage titer of each group was determined by the double-layer agar plate method. The MOI corresponding to the group with the highest phage titer was defined as the optimal MOI of vB-Efa1 [8]. Each experiment was set with 3 biological replicates, and the results were expressed as the average value.

2.6. Determination of One-Step Growth Curve

Logarithmic-phase Enterococcus faecalis culture (concentration: 1 × 10^8^ CFU/mL) was mixed with purified phage at the optimal MOI and incubated at room temperature for 3 min to ensure sufficient phage adsorption. Then 20 mL of sterile LB liquid medium was added, and the mixture was shake-cultured at 37 °C and 200 r/min for 3 h. Starting from 0 min of cultivation, 100 μL of sample was taken every 15 min, placed in a sterile centrifuge tube, centrifuged at 7000 r/min for 5 min, and the supernatant was collected and filtered with a 0.22 μm filter. The phage titer at each time point was determined by the double-layer agar plate method. A one-step growth curve was plotted with infection time as the abscissa and phage titer (lg PFU/mL) as the ordinate, and the latent period, lysis period, and burst size were calculated.

2.7. Determination of Phage Temperature Stability

One milliliter of purified phage solution was placed in sterile centrifuge tubes and incubated in constant-temperature water baths at 4 °C, 30 °C, 37 °C, 50 °C, 60 °C, and 70 °C for 1 h, respectively. After incubation, the samples of each experimental group were cooled to room temperature, and the phage titer was determined by the double-layer agar plate method. The group treated at 37 °C was used as the blank control, and the titer retention rate of phage in each temperature group was calculated (Titer retention rate = Titer of experimental group/Titer of control group × 100%). Each experiment was set with 3 biological replicates, and the results were expressed as the average value.

2.8. Determination of Phage pH Stability

SM buffers with pH gradients of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 were accurately prepared, and 900 μL of each buffer was added to sterile centrifuge tubes. Then 100 μL of purified phage solution was added to each tube, gently vortex-mixed for 30 s, and incubated at 37 °C for 1 h in a constant-temperature incubator. After incubation, the phage titer of each pH group was determined by the double-layer agar plate method, with the pH 7.0 group as the blank control, to analyze the effect of different pH conditions on phage activity stability. Each experiment was set with 3 biological replicates, and the results were expressed as the average value.

2.9. Determination of Phage Ultraviolet (UV) Stability

One milliliter of purified phage solution was added to sterile Petri dishes (the liquid level was controlled within 2 mm to ensure uniform UV irradiation). The Petri dishes were placed under a 30 W UV lamp at a distance of 30 cm for irradiation treatment, with irradiation times set to 0, 10, 20, 30, 40, 50, and 60 min, and one group of samples was taken every 10 min. A dark control group (treated under the same conditions but wrapped with black cloth) was set simultaneously. After sampling, the phage titer at each time point was immediately determined by the double-layer agar plate method. The 0 min irradiation group (initial titer) was used as the control, and the titer retention rate of each irradiation group was calculated (Titer retention rate = Titer of experimental group/Titer of control group × 100%). Each experiment was set with 3 biological replicates, and the results were expressed as the average value to analyze the effect of UV irradiation duration on phage activity.

2.10. Phage Genome Sequencing and Functional Annotation

Purified phage with a titer of 1 × 10^8^ PFU/mL was collected under sterile conditions and sent to Benagen Biotechnology Co., Ltd. for whole-genome sequencing. Sequencing was performed on the Illumina NovaSeq 6000 platform; de novo assembly of genome sequences was carried out using ABySS 2.20 software, coding sequences (CDS) were predicted by GeneMarkS 4.17 software, and a genome circular map was drawn with the help of the PhageScope tool. Homologous sequence alignment analysis of the genome was conducted using the BLASTN tool (2.17.0) in the NCBI database. Meanwhile, the genome sequence was aligned with the Antibiotic Resistance Genes Database (ARDB) and Virulence Factor Database (VFDB) to verify the presence of antibiotic resistance genes, virulence genes, and toxin genes, and to evaluate its application safety.

2.11. Application of Phage in Poultry Product Processing Safety (Determination of Bacteriostatic Effect on Chicken Breast Meat)

Fresh chicken breast meat sold in the market without antibiotic addition, without pathogenic bacteria contamination, and with a qualified total bacterial count was selected. Under sterile conditions, it was cut into uniform pieces of 2 cm × 2 cm × 1 cm (each weighing approximately 4 g), placed in sterile Petri dishes, and surface-sterilized by UV irradiation for 30 min on each side. After verifying surface sterility by colony counting (a colony count < 10 CFU/g was considered qualified), 100 μL of logarithmic-phase avian-derived Enterococcus faecalis culture was evenly spread on the surface of each chicken breast piece to achieve an initial bacterial concentration of 1 × 10^3^ CFU/g. The samples were incubated at 25 °C for 10 min to allow sufficient bacterial adsorption, and then purified phage vB-Efa1 solution of the corresponding volume was added according to the multiplicity of infection (MOI) values of 100, 10, 1, 0.1, and 0.01. The solution was evenly spread with a sterile spreader to ensure full contact between phage and bacteria. Bacteria-inoculated chicken breast pieces added with the same volume of SM buffer (without phage) served as the blank control group. In addition, two quality control groups were set up: the negative control group consisted of chicken breast pieces without artificial inoculation of Enterococcus faecalis, which were surface-sterilized by UV and added with the same volume of SM buffer (without phage), to eliminate the interference of indigenous microorganisms in chicken breast and operational contamination; the positive control group consisted of chicken breast pieces artificially inoculated with Enterococcus faecalis (initial bacterial concentration 1 × 10^3^ CFU/g) without adding phage or SM buffer, but only the same volume of sterile normal saline, to verify the normal growth ability of the target bacteria in chicken breast and ensure the effectiveness of the experimental system. 3 biological replicates set for each group. All samples were divided into two groups: one group was statically cultured in a 25 °C constant-temperature incubator in the dark (simulating room-temperature processing and temporary storage), and the other group was stored in a 4 °C refrigerator (simulating low-temperature storage). Samples were taken at 3 h, 6 h, 9 h, and 12 h, respectively. One gram of sample was aseptically weighed and placed in a homogenization bag containing 9 mL of sterile normal saline, homogenized at 8000 r/min for 1 min to prepare a 10^−1^ dilution, and then serially diluted 10-fold. One hundred microliters of the appropriate dilution was spread on LB agar plates, inverted and cultured at 37 °C for 24 h, and colony counting was performed to calculate the number of viable Enterococcus faecalis in chicken breast meat at each time point (unit: CFU/g). The results were expressed as “mean ± standard deviation” and subjected to significance analysis of bacteriostatic effect.

2.12. Data Processing

All experimental data were derived from three independent biological replicates (n = 3) to ensure experimental reproducibility. Raw data were sorted and organized using WPS 2025 software. Subsequently, Origin 2023 software was employed for data visualization (including titer change curves, bacteriostatic effect curves, and stability profile plots) and statistical analysis. Normality distribution was verified prior to parametric statistical testing. Differences in phage titer under varying temperatures, pH values, and multiplicity of infection (MOI) conditions were analyzed via two-way analysis of variance (two-way ANOVA) coupled with Tukey’s post hoc test. Statistical significance was defined as p < 0.05, and p < 0.01 indicated highly significant differences. Data variability was reported as the mean ± standard deviation (SD) across all figures, tables, and the main text. These modifications ensure the rigor, reliability, and intuitive presentation of the experimental results.

3. Results

3.1. Morphological Characteristics of the Phage

As shown in Figure 1A, phage vB-Efa1 formed plaques with a diameter of approximately 1 mm and no halos on the double-layer agar plates. As exhibited in Figure 1B, transmission electron microscopy (TEM) observation revealed that the isolated phage vB-Efa1 presented a typical siphovirus morphology: the head was a regular polyhedral structure, and the tail was slender and connected to the head. The average values of its morphological and dimensional parameters were as follows: head diameter of 62.25 nm, tail length of 253.31 nm, and tail width of 7.62 nm. These dimensional characteristics fall within the typical morphological range of the Siphoviridae family.

3.2. Optimal Multiplicity of Infection (MOI)

As shown in Figure 2, when MOI = 0.1, the titer of phage vB-Efa1 reached the highest, which was 8.87 lg PFU/mL. There was no significant difference in titer between the groups with MOI = 0.1 and MOI = 0.01.

3.3. One-Step Growth Curve

As shown in Figure 3 and the corresponding time-titer data, the proliferation kinetic characteristics of phage vB-Efa1 were as follows: It was in the early latent period from 0 to 15 min post-infection, with the titer stably maintained at 2.71~2.75 lg PFU/mL. During this stage, the phage completed adsorption, invasion, and preparation for progeny particle synthesis; at 30 min, the titer increased to 3.6 lg PFU/mL, marking the entry into the lysis period. Subsequently, the titer continued to rise, reaching 5.88 lg PFU/mL at 180 min, which was an increase of approximately 3.17 lg PFU/mL compared with the initial titer. The experimental data within 150 min reflected the stage-specific lytic release behavior of the phage, consistent with the one-step growth curve characteristics.

3.4. Phage Temperature Stability

This study analyzed the titer stability of phage vB-Efa1 at different temperatures (4 °C, 25 °C, 37 °C, 50 °C, 60 °C, 70 °C) over 1060 min (Figure 4). The results are shown in the figure: Under 37 °C and 25 °C, the phage titer remained at a high and stable level throughout the experiment. The titer of the 37 °C group was stable in the range of 8.438.47 lg PFU/mL, while that of the 25 °C group was maintained at 8.268.32 lg PFU/mL without obvious fluctuations; the titer also remained relatively stable at 4 °C, with only slight fluctuations within 7.417.70 lg PFU/mL. At 50 °C, the titer showed a slow downward trend, gradually decreasing from 7.35 lg PFU/mL at 10 min to 6.69 lg PFU/mL at 60 min, but still retained a certain level of activity. At 60 °C, the titer decreased more significantly, dropping from the initial 7.15 lg PFU/mL to 4.79 lg PFU/mL at 60 min. The phage exhibited extremely poor stability at 70 °C, with the titer decaying rapidly and dropping to 0 lg PFU/mL at 40 min, completely losing its activity. In summary, phage vB-Efa1 exhibited the highest stability in the range of 25~37 °C, maintained good activity at 4 °C, but was prone to inactivation at high temperatures of 70 °C and above.

3.5. Phage pH Stability

This study determined the activity stability of phage vB-Efa1 under different pH environments, and the results are shown in Figure 5: when pH was in the range of 14 and 1114, the phage lost its activity in such strongly acidic and strongly alkaline environments; when pH increased to 5, the titer recovered to 3.48 lg PFU/mL; in the pH range of 68, the titer was maintained at a high level (7.217.40 lg PFU/mL), with the peak titer (7.40 lg PFU/mL) achieved at pH 8; and when pH was 910, the titer decreased slightly (6.656.70 lg PFU/mL) but still maintained high activity. In conclusion, the suitable pH range for phage vB-Efa1 activity was 510, with the optimal pH range of 68. It had strong stability in neutral to weakly alkaline environments, which provided a pH adaptability basis for its application in practical environments (such as neutral water bodies and microbial communities).

3.6. Phage Ultraviolet (UV) Stability

This study determined the activity stability of phage vB-Efa1 under UV irradiation, and the results are shown in Figure 6: The initial titer (0 min) was 7.36 lg PFU/mL. With the extension of UV irradiation time, the phage titer showed a gradual downward trend: it decreased to 7.13 lg PFU/mL after 10 min of irradiation, 6.86 lg PFU/mL after 20 min, and 5.98 lg PFU/mL after 60 min. In summary, phage vB-Efa1 had a certain degree of UV stability and could maintain a certain level of activity after 60 min of UV irradiation, but the titer continued to decrease with the increase in irradiation time. This suggests that attention should be paid to controlling the UV exposure time of the phage in practical applications to reduce its activity loss.

3.7. Bioinformatics Analysis of Phage vB-Efa1

After quality control and filtering of the original sequencing data using FastP software v0.23.4 (removing reads with N content > 10% and low-quality base ratio > 50%), 1,225,460,654 bps of clean data were obtained (Figure 7). Sequencing quality assessment results showed that the Q20 rate reached 98.59% and the Q30 rate reached 94.62%, indicating excellent data quality, which provided a reliable data basis for subsequent genome assembly. De novo assembly was performed using Unicycler software (0.4.8), and the complete circular genome of phage vB-Efa1 was successfully obtained, with a genome length of 166,586 bp and a GC content of 35.46% (Figure 7). Gene structure annotation results showed that a total of 276 genes were predicted in the genome, with a total length of 158,239 bp, an average gene length of 573 bp, and the gene region accounting for 94.99% of the total genome length. Among them, it included 265 coding sequences (CDS) and 11 transfer RNAs (tRNA), and no ribosomal RNA (rRNA) was predicted. In addition, 30 pseudogenes were detected using Pseudofinder software (1.0), with a total length of 3834 bp and an average length of 127.8 bp, which were speculated to be non-functional sequence residues formed during evolution. Repeat sequence annotation results showed that the total length of repeat sequences in the genome was 734 bp, accounting for only 0.44%, including 2 Short Interspersed Nuclear Elements (SINE), 8 simple repeat sequences, 3 low-complexity repeat sequences, and 1 other type of repeat sequence. This is consistent with the typical characteristic that prokaryotic viral genomes generally have low repeat sequence content. Prophage element prediction results showed that there were 7 potential prophage sequences (named pp1–pp7) in the genome, distributed in different regions of the genome. Specific attL and attR attachment site sequences were detected in each prophage sequence, suggesting that integration events of prophage elements may have occurred in the phage genome during evolution.

To clarify the functional characteristics of CDS, the predicted CDSs were subjected to homologous alignment annotation with 8 general functional databases, with annotation coverage of 100% (all 265 CDS obtained functional annotations from at least one database): 100% of CDS could be annotated in the Nr, Uniprot, and Refseq databases; 46.79% of CDS in the Pfam database; 37.74% of CDS in the KEGG database; 22.64% of CDS in the GO database annotations; 8.68% of CDS in the TIGRFAMs database; and only 2.64% of CDS to the KEGG Pathway. Pfam domain annotation results showed that the top-ranked functional domains were mostly related to phage core structure assembly, including phage T4-like gp8 baseplate wedge subunit, gp7 baseplate wedge protein (containing helix domain, V domain, VI domain), gp6 baseplate structural protein (containing C-terminal domain), and PAAR motif protein. These domain characteristics were highly consistent with the morphological characteristics of the phage as a siphovirus observed by TEM earlier. GO functional classification results showed that gene functions were mainly enriched in three categories: cellular components (viral capsid, membrane structure), molecular functions (hydrolase activity, structural molecule activity, ATP binding), and biological processes (viral capsid assembly, translation regulation). KEGG Pathway annotation results mainly involved nucleotide metabolism (pyrimidine metabolism, purine metabolism) and genetic information processing (homologous recombination) pathways, providing important functional support for the life activities of the phage such as replication, assembly, and proliferation.

Safety-related functional annotation results showed that no antibiotic resistance genes were detected through resistance gene annotation analysis using ARDB and CARD databases; only one virulence factor-related sequence (ctg_00006) was annotated in the VFDB virulence factor database. This sequence encodes the type VI secretion system tip protein VgrG, with no obvious pathogenic risk indicated, suggesting that the phage has high safety when applied in environmental governance or biocontrol scenarios. Carbohydrate-Active enZymes (CAZy) annotation results showed that the genome contained 2 Glycoside Hydrolases (GHs) and 1 Glycosyltransferase (GTs), and no other types of carbohydrate-active enzymes such as polysaccharide lyases and carbohydrate esterases were detected. It is speculated that these enzymes may be involved in the degradation of host cell wall components, thereby assisting the phage in invading host cells. Five related sequences were annotated in the Pathogen-Host Interaction (PHI) database, among which 3 sequences may lead to reduced host virulence after mutation, and the other 2 sequences had no significant effect on host pathogenicity. In addition, signal peptide prediction results showed that 9 proteins contained signal peptide sequences; transmembrane protein prediction revealed that 19 proteins contained transmembrane helix structures, and a total of 8 secreted proteins were obtained after excluding transmembrane proteins; type III secretion system effector protein prediction results showed that 25 proteins may be effectors, including tail tube terminator protein, capsid vertex protein, short tail fiber protein gp12, and tail sheath protein. It is speculated that these effector proteins are involved in key processes such as phage recognition, adsorption, and invasion of the host. TCDB transporter database annotation results showed that some genes were involved in the regulation of membrane transport functions; the genome circular map intuitively presented core characteristics such as gene distribution, GC content, and GC skew, providing visual support for the correlation analysis of genome structure and function.

In summary, the genome of phage vB-Efa1 is a circular structure with excellent sequencing quality and complete assembly results; its gene functions are mainly enriched in pathways related to phage structural composition, replication and proliferation, and host interaction. No resistance genes were detected, and the risk associated with virulence factors is low.

3.8. Application of Phage vB-Efa1 in Chicken Breast Meat

This study analyzed the inhibitory effect of phage vB-Efa1 on Enterococcus faecalis on the surface of chicken breast meat at 4 °C and 25 °C under different multiplicities of infection (MOI), with the viable count of Enterococcus faecalis (CFU/g) used as the indicator to observe its dynamic changes. Under 4 °C refrigeration conditions, the viable count of Enterococcus faecalis on the surface of chicken breast meat in each MOI group was generally maintained at a low level without significant fluctuations, indicating that the phage exhibited a stable bacteriostatic effect: at 3 h, the viable count in the MOI = 1 group decreased to 3.21 lg CFU/g, while that in the other experimental groups (MOI = 100, 10, 0.1, 0.01) ranged from 3.35 to 3.68 lg CFU/g, and the count in the blank control group (added with SM buffer) was 4.89 lg CFU/g; at 9 h, the viable count in the MOI = 100 group slightly increased to 3.72 lg CFU/g, while the counts in the other experimental groups fluctuated slightly but were all significantly lower than that in the control group; by 12 h, the viable count of Enterococcus faecalis in all experimental groups stabilized at 3.40~3.85 lg CFU/g, while that in the control group increased to 5.62 lg CFU/g, with a significant difference between groups (Figure 8).

Under 25 °C room temperature conditions, the bacteriostatic effect of the phage in each MOI group was more significant, and the viable count of Enterococcus faecalis showed an obvious downward trend over time, with consistent change patterns among different MOI groups: At 3 h, the viable count in each experimental group ranged from 3.52 to 4.10 lg CFU/g, and that in the control group was 5.10 lg CFU/g. At 9 h, the viable count in all experimental groups decreased to below 3.0 lg CFU/g, among which the counts in the MOI = 0.1, MOI = 0.01 and MOI = 1 groups were close to 2.85 lg CFU/g, showing the optimal bacteriostatic effect. At 12 h, the viable count of Enterococcus faecalis in each experimental group was maintained at 2.70~3.15 lg CFU/g, while that in the control group increased to 6.05 lg CFU/g, indicating that the phage continued to exert its bacteriostatic effect. In summary, temperature is a key factor affecting the bacteriostatic effect of phage vB-Efa1 in chicken breast meat—the phage showed stronger bacteriostatic activity at 25 °C, which could significantly reduce the viable count of Enterococcus faecalis, while it could stably inhibit bacterial proliferation and maintain a low bacterial load at 4 °C. Within the experimental range, different MOI values had no significant effect on the bacteriostatic effect of the phage, and its bacteriostatic capacity was not significantly regulated by the multiplicity of infection.

4. Discussion

Enterococcus faecalis, a common opportunistic pathogen in the food chain, has seen the spread of its multidrug-resistant strains in poultry breeding and poultry product processing chains, posing a challenge to traditional prevention and control systems. Thus, developing targeted and efficient biological control technologies has become a research focus [9]. Phages, with advantages such as targeted lysis, no interference with beneficial microflora, and no chemical residues, are ideal biological agents to replace antibiotics for controlling avian-derived foodborne pathogens [10]. In this study, a virulent phage vB-Efa1 targeting avian-derived Enterococcus faecalis was isolated from the sewage of a poultry slaughterhouse. Systematic analysis of its biological characteristics, whole-genome decoding, and bacteriostatic verification in chicken breast meat matrix was conducted to clarify its control potential and molecular mechanism, thereby providing high-quality resources and theoretical support for preventing and controlling Enterococcus faecalis contamination in poultry products.

Morphological characteristics and lysis kinetics are core indicators for evaluating the control potential of phages. vB-Efa1 forms plaques with a diameter of approximately 1 mm and no halos. Transmission electron microscopy confirmed its typical morphology of the Siphoviridae family (head diameter: 62.25 nm, tail length: 253.31 nm), which is consistent with the characteristics of most reported Enterococcus faecalis siphophages [11]. The absence of halos suggests that it may lack genes encoding extracellular hydrolases, and its lysis depends on host cell rupture during progeny release [12]. Determination of the one-step growth curve and optimal MOI showed that vB-Efa1 had a latent period of approximately 30 min, with lysis and release lasting for at least 150 min. At the optimal MOI of 0.1, its titer reached 8.87 log_10_ PFU/mL, demonstrating a short latent period and strong continuous lytic capacity [13]. Whole-genome annotation indicated that it is enriched in genes related to DNA replication, nucleotide metabolism, and structural proteins, along with lysis-associated genes such as glycoside hydrolases and transmembrane proteins. These constitute a complete lysis system, which can enhance efficiency through auxiliary invasion and mediated lysis [14,15]. Compared with homologous phages with a latent period exceeding 40 min, its short latent period facilitates rapid inhibition of avian-derived Enterococcus faecalis proliferation, making it suitable for emergency control of poultry product contamination [16].

Environmental adaptability is crucial for the practical application of phages. vB-Efa1 maintains high activity within the range of 2537 °C and pH 68, and remains stable under 4 °C refrigeration, which is consistent with the requirements of room-temperature processing and low-temperature storage of poultry products [17]. Its inactivation at temperatures above 70 °C can be avoided by low-temperature spraying, and it retains activity after 60 min of ultraviolet irradiation, adapting to the disinfection process of poultry product processing [18]. It can tolerate a wide pH range of 510, making it more suitable for complex scenarios such as poultry product processing than homologous phages that only stabilize at pH 78 [19]. The mechanism of its acid-base tolerance is related to the conformational stability of capsid proteins, which are not easily denatured in neutral and weakly acid-base environments [20]. In summary, vB-Efa1 exhibits good stability under normal and low temperatures, making it suitable for laboratory preservation and poultry product processing scenarios. However, it is unstable under high temperatures and extreme acid-base conditions, and its tolerance needs to be optimized through formulation technologies such as microencapsulation [21].

Whole-genome analysis provides a molecular basis for the safety and functional specificity of vB-Efa1. Its circular genome (166,586 bp), GC content (35.46%), and 276 predicted genes are consistent with the typical characteristics of virulent Enterococcus faecalis phages [22]. Phylogenetic analysis showed that its core structural genes have high homology but genetic distance with known E. faecalis siphophages, providing a reference for its taxonomic status and phage diversity research [23]. Functional annotation revealed that core genes are involved in structural assembly, replication and proliferation, and host recognition, among which tail structural proteins are the key determinants of lytic spectrum [24]. vB-Efa1 contains 11 tRNA genes, which can reduce dependence on the host translation system, improve lysis efficiency and host adaptability, and provide a molecular explanation for its continuous lytic capacity [25,26]. Safety assessment showed no antibiotic resistance genes were detected, and only one virulence-related sequence with low pathogenic risk was identified, supporting its potential safety in poultry product control [27]. However, further in vivo toxicology tests, assessments of impacts on poultry intestinal microflora, endotoxin removal verification, and screening for horizontal gene transfer risks are required to ensure compliance with poultry product safety standards [28].

Bacteriostatic tests on chicken breast meat verified the practical application potential of vB-Efa1, with its bacteriostatic effect showing temperature dependence: at 25 °C room temperature, the phage significantly reduces the viable count of E. faecalis through continuous lysis, exhibiting strong bacteriostatic activity; under 4 °C refrigeration, it stably inhibits bacterial proliferation and maintains a low bacterial load [29]. This mechanism is related to the effect of temperature on the metabolism of avian-derived E. faecalis and phage proliferation kinetics—active host metabolism at 25 °C facilitates phage adsorption, replication, and assembly. Different MOI values have no significant impact on the bacteriostatic effect, suggesting that the dosage can be reduced to control costs and improve commercial feasibility [30]. This study fills the gap in the lack of poultry product matrix verification for avian-derived E. faecalis phages, providing direct data support for developing it into a special bacteriostatic agent for poultry product processing safety.

This study has limitations: First, the lytic spectrum is narrow, failing to cover avian-derived strains from different sources in the poultry product chain and multidrug-resistant strains. Second, the short-term observation of bacteriostatic tests on chicken breast meat cannot reflect the complexity of actual poultry product processing environments, and further verification is needed to confirm whether it meets the minimum poultry product safety standards. Third, the persistence of the phage in poultry products and its impact on product physicochemical properties were not explored [31,32,33]. Future research should: Expand the host range, focus on avian-derived drug-resistant strains, and construct composite preparations or expand the lytic spectrum through genetic engineering; Verify its efficacy under real poultry product processing conditions and in multiple poultry product matrices, and optimize stability through formulation technologies; Improve the safety assessment system, focus on screening potential risks to poultry breeding and poultry product quality, and promote practical transformation and application.

5. Conclusions

The virulent avian-derived E. faecalis phage vB-Efa1 isolated in this study possesses excellent biological characteristics (short latent period, continuous lytic capacity, optimal MOI of 0.1), good environmental adaptability (stable under normal and low temperatures and wide acid-base range, suitable for poultry product processing and storage scenarios), and high biosafety (no antibiotic resistance genes, only one virulence-related sequence with low pathogenic risk). It exhibits significant application potential in preventing and controlling E. faecalis contamination in poultry product processing safety (represented by chicken breast meat). This study enriches the resource pool of avian-derived E. faecalis phages, provides solid theoretical and technical support for developing new special biological bacteriostatic agents for poultry products, and contributes to the innovation and development of biological control technologies for poultry product processing safety.