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. 2026 Sep 16;13:101569. doi: 10.1016/j.crfs.2026.101569

Comprehensive characterization of Staphylococcus phage SAP_2 as a safe and effective biocontrol agent against methicillin-resistant Staphylococcus aureus

Dongxin Ma a, Shuaifeng Gu a, Yuqian Zhang a, Muhammad Shahzad Rafiq a, Ali Asif a, Jie Chen a, Mengran Xing a, Ziyi Li a, Sinuo Yang a, Yufeng Gu a, Pan Tao a,⁎, Haihong Hao a,b,⁎⁎
PMCID: PMC13601061  PMID: 42787317

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) contamination in the food chain poses a serious threat to public health, and bacteriophages have emerged as promising biocontrol agents. In this study, we isolated and characterized a broad-spectrum lytic Staphylococcus phage, SAP_2, from a livestock farm. SAP_2 showed lytic activity against 26 of 43 Staphylococcus aureus strains and 23 of 33 coagulase-negative staphylococci isolates in spot assays. It exhibited rapid adsorption, a short latent period (20 min), and a large burst size (173 PFU/cell), while remaining stable across pH 4-9, temperatures of 30-50 °C, ultraviolet irradiation, and chloroform treatment. Whole-genome analysis identified 219 open reading frames (ORFs) including eight predicted tail-associated proteins potentially involved in host recognition, with no virulence, antibiotic resistance, or lysogeny-associated genes. Phylogenetic analysis classified SAP_2 within the genus Kayvirus. In vitro, SAP_2 efficiently lysed MRSA at MOIs ≥ 0.01, compromised bacterial membrane integrity, and inhibited MRSA biofilm formation. In vivo, SAP_2 exhibited good biosafety and reduced bacterial burdens in MRSA-infected mice, with a numerically higher 7-day survival rate in the treatment group than in the model control group (66.67% vs. 33.33%). Furthermore, SAP_2 effectively reduced MRSA contamination in milk, with substantially greater antibacterial activity at 25 °C than at 4 °C. These findings support the potential of SAP_2 as a biocontrol candidate for controlling MRSA contamination in milk.

Keywords: MRSA, Broad-spectrum phage, Biofilm inhibition, Biosafety, Milk application

Graphical abstract

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Highlights

  • •

    Phage SAP_2 showed a relatively broad lytic spectrum among tested Staphylococcus strains including S. aureus and coagulase-negative staphylococci.

  • •

    Genomic safety and in vivo safety support SAP_2 as a biocontrol agent.

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    SAP_2 effectively inhibited MRSA planktonic growth and reduced biofilm biomass formation.

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    SAP_2 reduced inflammatory responses in mice and reduced secondary MRSA contamination in pasteurized milk.

1. Introduction

Staphylococcus aureus (S. aureus) is one of the most common foodborne pathogens and ranks among the leading causes of foodborne outbreaks worldwide (Collaborators, 2022). In the dairy industry, this pathogen primarily causes clinical and subclinical mastitis, resulting in milk contamination during production and processing (Tenhagen et al., 2023; Son and Duc, 2024). S. aureus contamination can occur during multiple stages of dairy production and processing. A review of 186 studies from 2001 to 2024 found MRSA in 68.8% of dairy product studies and 73.8% of bulk tank milk studies (González-Machado et al., 2024). Methicillin-resistant S. aureus (MRSA), in particular, poses a substantial public-health concern because of its antimicrobial resistance and associated morbidity and mortality (Zhou et al., 2018). In addition to methicillin resistance, biofilm formation can further complicate the control of MRSA by promoting bacterial persistence and reducing susceptibility to antimicrobial treatment. These biofilms act as a physical barrier and create an altered microenvironment, significantly enhancing MRSA resistance to multiple antibiotics and facilitating the formation of persister cells (Kunz Coyne et al., 2024). Meanwhile, regulatory restrictions on antibiotic use in food products and concerns about residues from other chemical pesticides further promote the development of safe alternative biocontrol agents (Ma et al., 2021; Zhou et al., 2025).

Phages are increasingly considered as an alternative for reducing bacterial infections in the food industry. Phages offer low cost, high specificity, and excellent safety compared with conventional antimicrobial agents. Numerous studies have shown that bacteriophages could effectively control bacterial contamination in foods. Staphylococcus phage KMSP1 could significantly reduce MRSA viable counts in pasteurized milk and cheese (Kwak et al., 2023). A broad-spectrum Staphylococcus phage SapYZU15 reduced viable counts in milk (by 5.69 log10 CFU/mL) and pork (by 1.16 log10 CFU/mL) at 25 °C (Wen et al., 2023). In addition, phages can control contamination caused by other foodborne pathogens. A novel Salmonella phage vB_SalS-LPSTLL has successfully controlled Salmonella in milk, apple juice, chicken, and lettuce (Guo et al., 2021). However, most studies lack a comprehensive evaluation that simultaneously covers in vitro antibacterial assessment, in vivo safety evaluation, in vivo therapeutic efficacy, and their application in food products.

In this study, we isolated a broad-spectrum polyvalent bacteriophage, SAP_2, using methicillin-resistant Staphylococcus aureus (MRSA, HN20) as the indicator strain. We systematically evaluated its host range, biological characteristics, genomic features and safety, in vitro lytic activity, and phage-associated antibacterial effects against MRSA. We also assessed the biosafety and therapeutic efficacy of SAP_2 in vivo. We investigated its antibacterial potential against MRSA in a food substrate (milk), providing a foundation for its use as a biocontrol agent in food safety and antimicrobial therapy.

2. Materials and methods

2.1. Bacterial strains, antimicrobial sensitivity test and bacterial sequencing

The Staphylococcus strains used in this study were isolated from livestock and poultry farms in China. The standard strains ATCC 29213 and RN4220 were obtained from the American Type Culture Collection (ATCC) and the National Collection of Type Cultures (NCTC), respectively. MRSA HN20 was used as the indicator strain for the isolation and propagation of phage SAP_2. Detailed information on all bacterial strains used in this study is provided in Table S1. All bacterial strains were cultured in BHI (Brain Heart Infusion, Hope Biotech) broth at 37 °C with shaking at 200 rpm. Antimicrobial susceptibility testing was performed using the broth dilution method (European Committee for Antimicrobial Susceptibility Testing of the European Society of Clinical et al., 2003) according to the breakpoints established by the Clinical and Laboratory Standards Institute (CLSI). The antimicrobial resistance profiles of the S. aureus isolates are shown in Fig. S1. Genomic DNA was extracted and purified from the Staphylococcus isolates using the Bacterial/Fungal DNA Extraction Kit (Magnetic Beads; Majorbio) and subsequently sequenced by Majorbio (Shanghai, China). Multilocus sequence typing (MLST) was performed using the PubMLST database based on the final assembled sequences.

2.2. Bacteriophage enrichment, purification, and concentration

Sewage samples were collected from livestock farms in Hubei Province and pretreated by sequential filtration through 0.45-μm and 0.22-μm filters (Merck Millipore). The resulting sewage filtrate (20 mL) was then mixed with an equal volume of 2×BHI broth and MRSA HN20, followed by overnight incubation at 37 °C with shaking at 220 rpm. The mixture was subsequently centrifuged and filtered through a 0.22-μm filter, and the resulting filtrate was collected as the phage stock. The presence of phages was confirmed by a spot assay, as described previously (Han et al., 2013).

For phage purification, individual plaques were picked and transferred into 1.5 mL tubes containing 300 μL of SM buffer (200 mM NaCl, 16 mM MgSO4, 100 mM Tris-HCl, 0.02% Gelatin, pH 7.5, Biocomma). The tubes were incubated overnight at 4 °C. Plaque purification using the double-layer agar (DLA) method was repeated at least 3 times as described previously (Zhang et al., 2021). The purified phage was stored at 4 °C for subsequent experiments.

2.3. Transmission electron microscopy (TEM)

Dialysis membranes (1.4 kDa, Bio-Sharp) and ultrafiltration centrifugal tubes (Millipore 4 mL/50 kDa) were employed to purify and concentrate phage lysate (Bonilla et al., 2016; Rafiq et al., 2025). Phage morphology was examined using transmission electron microscopy (TEM). A copper grid (XP-CF200) was treated with a glow discharge mass spectrometer (PELCO easiGlow TM 91000, Ted Pella) for 10 s (15 mA of current). The resulting phage concentrate (∼109 PFU/mL) was applied to the treated grid and allowed to adsorb for 1 min. The grid was then washed 10 times with distilled water, followed by negative staining with phosphotungstic acid (2%, pH 7.0; Leagene Biotech) for 1 min. After drying, the grids were examined using a transmission electron microscope (Talos L120C G2, Thermo Fisher Scientific) at 120 kV.

2.4. Phage-host range determination

The host range of phage SAP_2 was determined through spot assay (Han et al., 2013). A total of 74 clinical isolates and two standard strains were tested (Table S1) (Ponsecchi et al., 2024). For all strains showing visible lysis in the spot assay, productive infection was further evaluated by efficiency of plating (EOP) using the double-layer agar method. Serially diluted phage suspensions were plated on each test strain, and plaque titers were determined. EOP was calculated as the phage titer on the test strain divided by that on the indicator strain HN20. EOP values were classified as high productivity (EOP≥0.5), moderate productivity (0.1≤EOP<0.5), low productivity (0.001≤EOP< 0.1), or inefficient plating (EOP<0.001).

2.5. Multiplicity of infection (MOI) assay

The phage and host bacterium HN20 (106 CFU/mL) were mixed at MOIs from 0.0001 to 100. After incubation for 5 h at 37 °C with shaking, the mixtures were centrifuged at 12,000 rpm for 5 min, and phage titer was determined by the DLA method as previously described. All experiments were conducted in triplicate.

2.6. One-step growth curve

The phage and host bacterium HN20 were mixed at an MOI of 0.1 and incubated at 37 °C for 20 min. The mixture was then centrifuged at 12,000 rpm for 10 min. The resulting pellet was resuspended in 10 mL of preheated (37 °C) BHI broth and incubated at 37 °C with shaking at 200 rpm. Subsequently, 200 μL of the sample was taken and filtered every 20 min, and the titer was checked by the DLA method. All experiments were conducted in triplicate. Burst size (PFU/cell) was calculated as the final phage titer at the plateau phase (PFU/mL) divided by the initial bacterial cell concentration (CFU/mL).

2.7. Phage adsorption assay

Phage adsorption was determined according to a previously described method with minor modifications (Rafiq et al., 2025). Briefly, phage suspension (106 PFU/mL) was mixed with host bacterium S. aureus (HN20) at an MOI of 0.01 and incubated at 37 °C. Samples were collected at different time points (0, 2, 4, 6, 8, 10, 20, and 30 min) and centrifuged at 10,000 rpm to pellet the bacterial cells. The unadsorbed phages in the supernatant were finally assessed by determining the titer by the DLA method. All experiments were conducted in triplicate.

2.8. Phage stability tests

For pH stability, phage (109 PFU/mL) was incubated in SM buffer at various pH levels (2-11) at 37 °C for 1 h. For thermal stability, phage was incubated at various temperatures (30, 40, 50, 60, 70, and 80 °C) for both 30 min and 60 min. For ultraviolet (UV) stability, phage suspension (109 PFU/mL) was exposed to UV light (254 nm, 15 W) at a distance of 40 cm for 0 min, 20 min, 40 min, and 60 min. For chloroform (CHCl3) sensitivity, phage was mixed into CHCl3 at different concentrations (1%, 2%, and 5% v/v) for 1 h. Phage titers were determined using the DLA method. All experiments were conducted in triplicate.

2.9. Whole genome sequencing and analysis

The concentrated phage (109 PFU/mL) was treated with DNase I and RNase A (both 100 μg/mL, Sangon Biotech, Shanghai, China). Phage DNA was then extracted using the phage DNA extraction kit (Norgen Biotek Corp) according to the manufacturer's instructions.

Whole-genome sequencing was performed on the DNBSEQ-T7 (MGITech, China) platform. Quality-filtered reads were assembled using Unicycler v0.5.0 (Wick et al., 2017). Open reading frames (ORFs) were then annotated using DFAST v.1.6.0 (Tanizawa et al., 2018), DNA Master (Pope and Jacobs-Sera, 2018), and HHpred (Soding et al., 2005). tRNA genes, antibiotic resistance genes, and virulence factors were predicted using tRNAscan-SE (Lowe and Eddy, 1997), ResFinder (Florensa et al., 2022), and the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/), respectively. A circular genomic map of phage SAP_2 was generated using Proksee (https://proksee.ca).

For phylogenetic analysis, the major capsid protein of phage SAP_2 was used as the query for BLASTp searches, and homologous protein sequences were retrieved for further analysis. Sequences were aligned using MEGA v.10.1.8. A maximum-likelihood phylogeny was inferred using MEGA v.10.1.8. Branch support was assessed using 1,000 bootstrap replicates. The resulting tree was visualized and annotated using iTOL. Additionally, a genome-wide proteomic tree was generated using the Viptree server (v4.0; https://www.genome.jp/viptree/). The complete genome of SAP_2 was uploaded as the query, and related reference phages were selected based on genome-wide similarity, yielding 153 phage genomes representing 16 genera. Pairwise genome-wide similarities were calculated using tBLASTx, and the proteomic tree was constructed by BIONJ based on genomic distances derived from normalized similarity scores (1-S_G), using the default Viptree settings.

The Vclust tool (https://afproject.org/vclust/) was used to generate a heatmap illustrating the genomic nucleotide sequence similarity between phage SAP_2 and other sequenced phages. Comparative genomic analysis was performed using Easyfig v.2.2.5, focusing on Staphylococcus phage SAP_2, Staphylococcus phage (RuSa1), and the top hits identified by BLASTn analysis.

2.10. Bacterial cell lytic assay

To evaluate phage lytic activity, host HN20 was mixed with phage at varying MOIs (10, 1, 0.1, 0.01) in a 96-well plate. The control group received an equal volume of BHI broth instead of phage. The plate was incubated at 37 °C with shaking at 180 rpm for 10 h using a fully automatic growth curve analyzer (Bioscreen C, Oy Growth Curves Ab Ltd). OD600 was measured every 30 min. All experiments were performed in triplicate.

2.11. Flow cytometry detection

To assess phage-induced changes in bacterial membrane integrity, host HN20 was mixed with phage at an MOI of 0.01 and incubated at 37 °C. An equal volume of BHI broth was added to the bacterial control cells instead of phage. After 24 h of incubation, the bacterial cells were collected, and the supernatant was discarded. The cells were washed three times with phosphate-buffered saline (PBS). Bacterial membrane integrity was assessed using an Annexin V-FITC/PI Apoptosis Detection Kit (Meilun Biotech) according to the manufacturer's instructions. Briefly, 400 μL of 1×binding buffer was added to the samples, followed by 5 μL of FITC and 10 μL of PI. The samples were incubated for 15 min, after which an additional 400 μL of 1×binding buffer was added. The samples were subsequently analyzed by a flow cytometry (Cytek).

2.12. Scanning electron microscopy (SEM) and TEM observation of bacteria

For scanning electron microscopy (SEM), phage SAP_2 was mixed with bacterial culture (106 CFU/mL) at an MOI of 0.01 and incubated at 37 °C. Untreated bacterial cells served as the control. After incubation, the bacterial cell pellet was collected by centrifugation, washed three times with PBS, and treated with 2.5% glutaraldehyde (1 mL). The samples were resuspended, and fixed overnight at 4 °C. After fixation, the samples were subjected to gradient dehydration using ethanol solutions at 30%, 50%, 70%, 80%, 90%, 100%, and 100% for 15 min at each concentration. The samples were centrifuged to remove the supernatant and then freeze-dried. The dried samples were mounted on a conductive adhesive on the copper bench and coated with a metal film by an ion sputter instrument. Bacterial surface morphology was examined using a field-emission scanning electron microscope (FE-SEM, SU, TECHCOMP, Japan) (Gordillo Altamirano et al., 2021).

For bacterial TEM analysis, bacterial precipitates from the phage-treated and control groups were collected, resuspended, and fixed for 4 h with 2.5% glutaraldehyde at 25 °C. Ultrathin sections were prepared and examined by TEM.

2.13. Biofilm inhibition assay

Crystal violet staining was used to evaluate the ability of phage to inhibit biofilm formation (Hu et al., 2022). Phage SAP_2 was mixed with the bacterial solution at an MOI of 0.01 and incubated at 37 °C for 24 h. Control wells received an equal volume of bacterial culture without phage. After incubation, the biofilms were washed three times with PBS, and stained with crystal violet (1%) for 20 min. The bound crystal violet was dissolved in glacial acetic acid (analytical grade) and absorbance was measured at 570 nm using a spectrophotometer (UQuant, Olympus, Japan). All experiments were performed in triplicate.

2.14. Experimental design of the in vivo study

Healthy female C57BL/6 mice (5-week-old) were adapted to the environment with a suitable temperature (20-24 °C), relative humidity (50-60%), and a 12-h light/darkness cycle for 7 days. For the safety assessment, mice were randomly divided into two groups (n = 6): normal control (NC) and SAP_2. Mice in the SAP_2 group were intraperitoneally injected with Staphylococcus phage SAP_2 (2 × 108 PFU/mouse) for seven consecutive days, and the NC group with an equal volume of SM buffer (pH 7.5). Mouse behavior and clinical signs were monitored daily. On day 7, the mice were anesthetized, and the blood samples were collected from the orbital veins. Serum was separated and evaluated using an automatic biochemical analyzer. The kidneys and liver were collected, weighed, and fixed in 4% paraformaldehyde. The tissues were subsequently subjected to H&E staining for histopathological examination.

For the efficacy assessment, mice were randomly divided into three groups: Normal control (NC), Model control (M), and SAP_2 treatment (ST). For each group, six mice were used for the 7-day survival analysis and another six mice were used for terminal sample collection on day 7 (n = 12 per group in total). MRSA HN20 was adjusted to 2 × 108 CFU/mL and intraperitoneally injected (100 μL) into each group except NC. After 2 h, the ST group received Staphylococcus phage SAP_2 (2 × 108 PFU/mouse) intraperitoneally once daily for three consecutive days, while the M and NC groups received an equivalent volume of PBS according to the same administration schedule. The six mice assigned to survival analysis were monitored for 7 consecutive days. The other six mice in each group were euthanized on day 7 for terminal sample collection. Blood samples and organ tissues were collected. Serum was used to determine TNF-α and IL-6 levels using an enzyme-linked immunosorbent assay (ELISA) kit. The heart, liver, spleen, lung, and kidney tissues were collected for organ-index determination, qPCR-based bacterial burden analysis, and histopathological examination.

All mice were obtained from the Laboratory Animal Center of Huazhong Agricultural University. All experiments were approved by the Animal Ethics Committee of Huazhong Agricultural University and conducted in accordance with institutional guidelines for the care and use of experimental animals (Ethical ID Number: 202603120004).

2.15. qPCR-based estimation of bacterial burden in organ tissues

For qPCR-based bacterial burden estimation, approximately 10 mg of each tissue was homogenized in 0.5 mL of sterile PBS, and genomic DNA was extracted from 200 μL of homogenate using the FastPure Blood/Cell/Tissue/Bacteria DNA Isolation Mini Kit (DC112-01; Vazyme) according to the manufacturer's instructions. qPCR targeting the S. aureus nuc gene was performed in a 20-μL reaction containing 10 μL of 2 × qPCR master mix, 0.4 μM each primer, 2 μL of DNA template, and nuclease-free water. Primer sequences and cycling conditions are provided in Fig. S6.

The standard curve was generated using bacterial suspensions processed through the same DNA extraction and qPCR procedures as tissue samples. The standard-curve procedure was independently repeated and yielded comparable regression parameters, supporting the reproducibility of the qPCR assay. Bacterial concentrations were estimated from the standard curve (Cq = −3.771 × log10 (C) + 45.45), where C represents CFU-equivalents/mL. The qPCR-estimated CFU-equivalent S. aureus burden in each tissue was calculated as CFU-equivalents/g = C × D × V/m, where D is the dilution factor, V is the total homogenization volume (mL), and m is the tissue mass (g). Results were expressed as log10 CFU-equivalents/g tissue.

2.16. Staphylococcus phage SAP_2 application in milk

Skim milk was used to evaluate the antibacterial activity of phage SAP_2 against MRSA (HN20) at 4 °C and 25 °C. Skim milk powder was purchased from the supermarket in Wuhan, China. Skim milk (10% w/v) was freshly prepared and sterilized before bacterial inoculation. Milk samples (9 mL) were inoculated with 1 mL of MRSA HN20 suspension (107 CFU/mL) and supplemented with phage SAP_2 at an MOI of 10 or 0.01. Samples contaminated with bacteria but without phage served as controls. All groups were incubated at 4 °C and 25 °C (room temperature) for 24 h, with sampling at 0, 3, 6, 12, and 24 h; 10-fold serial dilutions were prepared in phosphate-buffered saline (PBS) to determine viable bacterial counts. All experiments were performed in triplicate.

2.17. Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0.1. Data from experiments with at least three independent replicates are presented as mean ± standard deviation (SD), unless otherwise specified in the figure legends. A significance level of p < 0.05 was employed for all statistical analyses.

3. Results and discussion

3.1. Phage isolation and morphology

The S. aureus strain HN20 isolated from livestock showed a multidrug-resistant phenotype and was identified as MRSA (Fig. S1). Using this strain as the host, we isolated a virulent phage (SAP_2) from wastewater collected from livestock farms in Hubei Province. The purified phage SAP_2 formed clear, round plaques on the host bacterial lawn, with a diameter of 1.00 ± 0.5 mm (Fig. 1A). TEM observation showed that phage SAP_2 possessed an icosahedral head of approximately 164 ± 5.66 nm in diameter and a long contractile tail of 204 ± 5.43 nm in length (Fig. 1B). Based on its morphology, SAP_2 exhibits a myovirus-like morphology characterized by a long contractile tail. Genome-based taxonomic analysis further assigned SAP_2 to the genus Kayvirus within the family Herelleviridae and class Caudoviricetes.

Fig. 1.

Fig. 1

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Morphological and biological characterization of phage SAP_2. (A) Plaque morphology of phage SAP_2 on a BHI double-layer agar plate. (B) Transmission electron microscopy showing the morphology of phage SAP_2 at 120 kV. Scale bar represents 200 nm. Images were taken at SA 57,000× magnification. (C) Phage yield following infection at different initial multiplicities of infection. (D) One-step growth curve of phage SAP_2. (E) Phage adsorption assay. The phages were added to the host culture at a MOI of 0.01. (F) The stability of phage SAP_2 at different pH levels. No viable phages were detected at pH 2, 3, 10, or 11; these values were therefore not plotted on the logarithmic y-axis. (G) The stability of phage SAP_2 at different temperatures. (H) Phage sensitivity to the ultraviolet. (I) Phage sensitivity to chloroform. Data are presented as mean ± SD (n = 3). One-way ANOVA with Tukey's multiple-comparison test was used for C, F, H, and I, and two-way ANOVA with Tukey's multiple-comparison test was used for G. Different letters indicate significant differences (p < 0.05).

3.2. Host range of phage SAP_2

Table S1 summarizes the host range of phage SAP_2 determined by spot assay and EOP analysis. In the initial spot assay, SAP_2 produced visible lysis on 26 of the 43 S. aureus strains and 23 of the 33 coagulase-negative Staphylococcus (CoNS) strains, corresponding to an overall spot-assay lysis rate of 64.47%. To distinguish productive infection from spot clearing, EOP was subsequently determined for all spot-positive strains (Table S1; Fig. S2). Productive infection was confirmed for all spot-positive S. aureus strains tested, although plating efficiency varied among strains. In contrast, the spot-positive CoNS isolates showed a broader range of EOP values, including several isolates with EOP<0.001, indicating that spot clearing did not necessarily correspond to efficient productive infection. Notably, the spot-positive panel included ten MRSA isolates, 16 methicillin-resistant CoNS, and two standard strains. Together, these results demonstrate that SAP_2 has a relatively broad lytic spectrum within Staphylococcus, while the efficiency of productive infection is strain dependent.

3.3. Phage characterization

The biological characteristics of bacteriophages are crucial for determining whether they possess properties suitable for specific applications. Key phage parameters, including phage MOI, latent period, burst size, and stability, can all provide useful information from phage studies (Gill and Hyman, 2009). Among the tested initial MOIs, an MOI of 0.01 yielded the highest phage titer after 5 h of co-incubation (2.7 × 109 PFU/mL; Fig. 1C). This initial MOI was lower than the values reported to yield the highest progeny phage titers for phage SPB (MOI = 1) (Ma et al., 2025a, Ma et al., 2025b) and vB_SauH_SPJ2 (MOI = 10) (Zhou et al., 2025). The one-step growth curve revealed that SAP_2 showed a latent period of 20 min, followed by a rise period of about 100 min, with average burst size of 173 PFU/cell (Fig. 1D). Its burst size surpassed vB_SauS-phiIPLA35 (27 PFU/cell), vB_SauS-phiIPLA88 (45 PFU/cell) (García et al., 2009), and SauPS-28 (25 PFU/cell) (Zhao et al., 2024). This result indicates that SAP_2 is more efficient at intracellular replication, assembly, and lysis. The adsorption kinetics showed that approximately 91.5% of phage SAP_2 adsorbed to host cells within 30 min (Fig. 1E).

We evaluated the stability of phage SAP_2 under different pH levels, temperatures, UV exposure times, and chloroform (CHCl3) concentrations. SAP_2 showed stable activity at pH 4-9. However, its titer rapidly decreased under acidic or alkaline conditions (Fig. 1F). When heated to 60 °C, the titer of SAP_2 decreased rapidly, and no viable phages were detected at 80 °C (Fig. 1G). Its stability at pH 4-9 and temperatures of 30-50 °C was comparable to phage LTH02 (Ma et al., 2025a, Ma et al., 2025b), enabling it to adapt to various environmental conditions in potential application scenarios. In addition, phage SAP_2 remained stable during UV exposure for up to 60 min and after 1-h exposure to 1-5% chloroform (CHCl3) (Fig. 1H and I). These results show that SAP_2 is highly stable across a relatively broad range of tested conditions, but sensitive to extreme pH and high temperature.

3.4. Genomic characteristics of Staphylococcus phage SAP_2

The complete genome of phage SAP_2 was assembled as a linear double-stranded DNA molecule of 142,111 bp with a GC content of 30% (Fig. 2). A total of 219 open reading frames (ORFs) and three tRNA genes were predicted. Of the 219 ORFs, 88 were assigned putative functions based on homology searches (Table S2). The annotated ORFs are divided into five modules: morphogenesis, DNA replication & metabolism, lysis, additional functions, and hypothetical proteins. Among these, SAP_2 was predicted to encode eight tail-associated proteins, some of which may participate in host recognition and thereby contribute to its host range (Leprince and Mahillon, 2023). However, their receptor-binding functions and the corresponding bacterial receptors remain to be experimentally validated. Notably, phage SAP_2 did not carry genes encoding integrase or repressor proteins, thereby supporting a lytic lifestyle. This characteristic is consistent with most Staphylococcus phages isolated from food, including phiIPLA88 (García et al., 2009) and SAP-2 (Son et al., 2010), and is an important prerequisite for using phages in food-related applications. In addition, SAP_2 lacks identified virulence or antibiotic resistance genes, providing additional genomic support for its safety assessment (Koutsoumanis et al., 2023).

Fig. 2.

Fig. 2

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Genome map of phage SAP_2 with functional annotation showing absence of virulence, toxin, and antibiotic resistance genes. The genomic map of phages was created by Proksee. Different modules were colour-coded according to the functions of ORFs.

BLASTn analysis revealed that phage SAP_2 shared 98.96% nucleotide identity (95% coverage) with RuSa1 and 94.43% nucleotide identity (92% coverage) with CUB-B. Whole-genome comparisons of SAP_2, RuSa1, and CUB-B showed broadly similar genomic organization, with differences mainly involving local gene arrangement and hypothetical-protein regions (Fig. 3A). Whole-genome nucleotide similarity between phage SAP_2 and other related staphylococcal phages was assessed using Vclust (Fig. 3B). The heatmap revealed that phage SAP_2 shared the highest similarity (tANI = 93.91%) with Staphylococcus phage RuSa1. We constructed a maximum-likelihood phylogenetic tree based on the amino acid sequences of the major capsid protein of phage SAP_2 and related phages, including the well-characterized reference phages Twort, K, G1, JD007, GH15, and P108. SAP_2 clustered within the Kayvirus clade together with these reference phages (Fig. 3C). A complementary Viptree analysis based on genome-wide proteomic similarity likewise placed SAP_2 within the broader evolutionary context of related staphylococcal phages (Fig. S3).

Fig. 3.

Fig. 3

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Genomic and phylogenetic analysis of phage SAP_2. (A) Whole-genome comparison of SAP_2 with the closely related Staphylococcus phages RuSa1 and CUB-B generated using Easyfig v2.2.5. Sequence similarity is represented by the intensity of gray shading, and ORFs are colored according to predicted function. (B) Heatmap showing pairwise intergenomic nucleotide similarity between SAP_2 and other related Kayvirus phages, calculated using Vclust. (C) Maximum-likelihood phylogenetic tree based on major capsid protein sequences from SAP_2 and related phages, including the well-characterized reference phages Twort, K, G1, JD007, GH15, and P108. The tree was generated using MEGA v.10.1.8 with 1000 bootstrap replicates. SAP_2 is highlighted in red.

3.5. In vitro antibacterial activity and anti-biofilm activity of phage SAP_2 against MRSA

As shown in Fig. 4A, the control group exhibited rapid bacterial growth. In contrast, the phage-treated groups (MOI ≥ 0.01) showed progressively decreasing growth rates with increasing MOI, and the growth curves became increasingly flattened at higher MOI values. At high MOI, bacterial growth was nearly arrested early and showed only minimal increase later, indicating a strong, MOI-dependent inhibitory effect of multiplicity of infection on host bacterial accumulation.

Fig. 4.

Fig. 4

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Assessment of antibacterial activity in vitro. (A) Growth curves of MRSA HN20 treated with phage SAP_2 at different MOIs (10, 1, 0.1, 0.01). Data represent mean ± SD from three independent experiments. (B) Flow cytometry analysis of membrane integrity. Representative dot plots and quantification of Annexin V/PI staining populations in MRSA HN20 after phage SAP_2 treatment. (C) SEM observation of bacterial surface morphology after phage treatment. Scale bars: 2.00 μm. (D) TEM images of MRSA HN20 after phage treatment. Scale bars: 500 nm. (E) Inhibitory effect of SAP_2 on MRSA HN20 biofilm formation. Bars indicate the mean absorbance at 570 nm. Data are presented as mean ± SD (n = 3), and analyzed using an unpaired two-tailed Student's t-test. Different letters indicate significant differences (p < 0.05).

In addition, phage SAP_2 markedly suppressed planktonic MRSA growth and significantly inhibited MRSA biofilm formation. Flow cytometry analysis showed that MRSA lost cell membrane integrity after phage treatment, and the propidium iodide (PI)-positive population increased from 0.035% to 52.12%, an increase of 52.09 percentage points compared with the control group (Fig. 4B), indicating a marked loss of membrane integrity. Electron microscopy further revealed phage-associated morphological damage to the bacterial cells. SEM revealed numerous phage particles adsorbed to the surface of treated bacterial cells (Fig. 4C; Fig. S4), together with shrunken and collapsed cells showing apparent surface damage (Young, 2014). Concurrently, TEM showed that SAP_2 replicates and assembles inside bacterial cells, with cell wall rupture and leakage of cytoplasmic contents (Fig. 4D; Fig. S5). These effects likely result from the synergistic activity of phage-encoded holin, endolysin, and other proteins involved in cell lysis (Young, 1992). Notably, SAP_2 inhibited MRSA HN20 biofilm formation by 52.5% compared with the untreated control (Fig. 4E). These results demonstrate that SAP_2 can inhibit biofilm formation and reduce the biomass of MRSA biofilms under the tested conditions. Compared with previously characterized MRSA phages, such as SA-W2 and MRSA-W3 (Chopjitt et al., 2026), which showed biofilm inhibition rates of approximately 22-45%, SAP_2 exhibits substantial activity against MRSA biofilms.

3.6. The biosafety evaluation of phage SAP_2 in mice

To assess the biosafety of phage SAP_2, a mouse model was used for phage injection over 7 consecutive days (Fig. 5A). No mortality or obvious signs of illness (such as weight loss, piloerection, lethargy) were observed in either group during the 7-day observation period. At the end of the experiment, the liver and kidneys were collected and weighed. As shown in Fig. 5B and C, there were no significant differences in the liver index (liver weight/body weight) or kidney index (kidney weight/body weight) between the phage-treated and control groups (4.24 ± 0.23% vs. 4.68 ± 0.44% for liver; 1.16 ± 0.04% vs. 1.20 ± 0.03% for kidneys, p > 0.05). Additionally, we measured serum levels of liver function markers (alanine aminotransferase, ALT; aspartate aminotransferase, AST; alkaline phosphatase, ALP) and kidney function markers (creatinine, CREA; urea, UREA; uric acid, UA). As shown in Fig. 5D and E, no statistically significant differences were observed between the SAP_2 and NC groups for any of these parameters (p > 0.05 for all). Liver and kidney tissues were collected for hematoxylin and eosin (H&E) staining. Representative sections are shown in Fig. 5F. In the NC group, hepatocytes exhibited normal morphology with well-defined nuclei and sinusoids. In contrast, the kidney showed intact glomeruli and tubules without inflammation or necrosis. The SAP_2 group displayed comparable histoarchitecture, with no evidence of vacuolation, inflammatory infiltration, fibrosis, or necrosis, providing no histopathological evidence of obvious liver or kidney injury under the tested conditions. These results demonstrate that no obvious short-term toxicity was detected in SAP_2-treated mice under the tested conditions.

Fig. 5.

Fig. 5

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In vivo biosafety of phage SAP_2. (A) Experimental design for safety assessment of the phage SAP_2. Liver (B) or kidney (C) to body weight ratio (%) of mice after SM or phage treatment for seven days. (D) Serum biochemical analysis of liver function, including ALT, AST and ALP. (E) Serum biochemical analysis of kidney function, including CREA, UREA and UA. (F) Liver and kidney histopathology in phage-treated mice. Scale: 200 μm. Box-and-whisker plots (B, C) show the median, interquartile range, and minimum-to-maximum values; violin plots (D, E) show the distribution of individual values, with the horizontal line indicating the median. No significant differences were detected between groups (unpaired two-tailed Student's t-test, n = 6, p > 0.05).

3.7. Therapeutic efficacy of phage SAP_2 in mice with MRSA infection

To evaluate the therapeutic potential of phage SAP_2, a mouse model was used for MRSA infection, and phage treatment was administered for 3 consecutive days (Fig. 6A). For survival analysis, the observed 7-day survival rates were 66.67% in the ST (SAP_2 treatment) group and 33.33% in the M (Model control) group (Fig. 6B). The survival curves did not differ significantly between these groups (log-rank [Mantel-Cox] test, p = 0.279). In the mice used for terminal analyses, serum and organ tissues were collected on day 7. TNF-α and IL-6 levels were significantly higher in the M group than in the NC group, consistent with the inflammatory response induced by MRSA infection. As shown in Fig. 6C, compared with the M (Model control) group, TNF-α was significantly lower in the ST group, indicating that SAP_2 treatment was associated with attenuation of the systemic inflammatory response. The IL-6 level after SAP_2 treatment was also significantly lower than the model infection group, indicating that SAP_2 treatment may mitigate acute inflammation and was associated with attenuation of the systemic inflammatory response (Fig. 6D). The organ (heart, liver, spleen, lung, and kidney) indices were significantly lower in the ST group than in the M group.

Fig. 6.

Fig. 6

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In vivo efficacy of phage SAP_2 in a mouse model of MRSA infection. (A) Experimental design for systemic infection mouse model and phage treatment. (B) Survival curves of mice monitored for 7 days after MRSA challenge and phage treatment. (C-D) Serum concentrations of TNF-α (C) and IL-6 (D) in the NC group, model control (M), and SAP_2-treated (ST) groups. (E) Organ-to-body-weight ratios (%) in the NC group, model control (M), and SAP_2-treated (ST) groups. (F) qPCR-estimated S. aureus burdens in organs (heart, liver, spleen, lung and kidney) of model control (M), and SAP_2-treated (ST) groups. (G) Histopathology of the heart, liver, spleen, lung and kidney from the NC, M, and ST groups. NC, normal control; M, model control (MRSA infection); ST, phage SAP_2 treatment. Survival curves (B) were compared using the log-rank (Mantel-Cox) test; data in C-E were analyzed by one-way ANOVA followed by Tukey's multiple-comparison test, and data in F by anCommentHighlight unpaired two-tailed Student's t-test (n = 6). Different letters indicate significant differences (p < 0.05).

Additionally, we assessed qPCR-estimated CFU-equivalent bacterial burdens in organ tissues (Fig. S6). The ST group showed significantly lower organ indices (Fig. 6E) and qPCR-estimated S. aureus burdens (Fig. 6F) than the M group. These findings indicate that phage SAP_2 treatment was associated with reduced inflammatory responses and lower tissue bacterial burdens in MRSA-infected mice. Histopathological examination further showed less severe infection-associated tissue alterations in the ST group than in the M group (Fig. 6G). Compared with the NC group and ST group, cardiomyocytes in the M group were disordered in arrangement, and myocardial fibers were dissolved. The liver showed steatosis, accompanied by marked inflammatory cell infiltration around the liver sinusoids and portal areas. The spleen's normal structure was destroyed, with diffuse and nodular lymphopoiesis. The normal alveolar structure was damaged and the alveolar septa were thickened. In the kidneys, scattered inflammatory cell infiltration or edema was observed. These results support the potential therapeutic activity of SAP_2 in vivo, although the improvement in 7-day survival did not reach statistical significance.

3.8. Application of phage SAP_2 for controlling MRSA in milk

The high prevalence of S. aureus contamination in milk and dairy products has become a prominent food safety concern (González-Machado et al., 2024). Although commercial milk generally undergoes pasteurization or sterilization before consumption, secondary bacterial contamination during subsequent stages, such as cooling, filling, packaging, storage, and distribution, remains a concern. At 4 °C, the control MRSA population increased slightly from 6.24 log10 to 6.43 log10 CFU/mL over 24 h. Treatments with SAP_2 at MOIs of 0.01 and 10 resulted in bacterial counts of approximately 6.15 log10 CFU/mL and 6.07 log10 CFU/mL, respectively. Thus, SAP_2 produced only a modest reduction of approximately 0.28-0.36 log10 CFU/mL relative to the untreated control at 24 h (Fig. 7A). In contrast, SAP_2 showed substantially stronger antibacterial activity at 25 °C. The viable bacterial counts in the MOI = 10 treatment groups were significantly reduced (p < 0.05), decreasing to approximately 4.88 log10 CFU/mL relative to untreated control at 24 h, with a reduction of 3.60 log10 CFU/mL (Fig. 7B). Together, these findings indicate that the antibacterial efficacy of SAP_2 in milk is strongly temperature dependent, with substantially greater activity at 25 °C than at 4 °C. Wu et al. observed that in a dairy product model, phage cocktails administered at high MOIs (≥10) more effectively suppressed Salmonella typhimurium growth at room temperature. In contrast, lower temperatures substantially impeded phage adsorption and replication dynamics (Wu et al., 2026). Furthermore, in the present study, the group treated with an MOI of 0.01 at 25 °C still showed measurable inhibitory effects (2.37 log10 CFU/mL reduction), suggesting that SAP_2 retains potential utility even at a low initial MOI. This observation is consistent with Waturangi et al., who reported that phage therapy can still maintain inhibitory thresholds in food matrices at a low initial MOI (0.01) (Waturangi et al., 2021). These findings highlight the potential of phage SAP_2 as a targeted biocontrol agent for combating MRSA.

Fig. 7.

Fig. 7

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Antibacterial activity of phage SAP_2 against MRSA (HN20) in milk at 4 °C (A) and 25 °C (B). Data were analyzed separately at each temperature using two-way ANOVA followed by Tukey's multiple-comparison test (n = 3). Different letters indicate significant differences among treatments at the same time point (p < 0.05).

3.9. Limitation of the study

Our study aimed to investigate the inhibitory effects of phage SAP_2 on food substrate (milk) against MRSA. We tested only one MRSA strain, limiting our assessment of phage SAP_2's broad-spectrum antibacterial activity across different MRSA isolates. In addition, the experimental conditions in this study covered only two conventional storage temperatures. Other food-processing conditions, such as acidic pH, high pressure, and pasteurization were not evaluated. Future studies should evaluate multiple MRSA strains and a broader range of food-processing and storage conditions.

4. Conclusion

In this study, we isolated and comprehensively characterized the lytic bacteriophage SAP_2 targeting methicillin-resistant Staphylococcus aureus (MRSA) through genomic, in vitro, in vivo, and food-matrix evaluations. Genomic analysis identified no known lysogeny-associated, virulence, or antibiotic resistance genes. The phage showed a relatively broad lytic spectrum among the tested Staphylococcus isolates and effectively inhibited MRSA biofilm formation. In vivo, SAP_2 showed no obvious toxicity in mice and reduced qPCR-estimated bacterial burdens and inflammatory responses following MRSA challenge. In milk, the phage showed strong temperature-dependent antibacterial activity, producing only a modest reduction at 4 °C but substantially greater reductions at 25 °C. Collectively, these findings support the potential of SAP_2 as a biocontrol candidate against MRSA, while further studies under broader food-processing and storage conditions will be required to establish its practical applicability.

CRediT authorship contribution statement

Dongxin Ma: Writing-original draft, Methodology, formal analysis. Shuaifeng Gu: writing-review & editing, data curation. Yuqian Zhang: investigation, conceptualization. Muhammad Shahzad Rafiq: investigation, visualization. Ali Asif: writing-review & editing. Jie Chen: investigation, formal analysis. Mengran Xing: investigation, Methodology. Ziyi Li: formal analysis. Sinuo Yang: methodology. Yufeng Gu: data curation. Pan Tao: supervision, project administration. Haihong Hao: project administration, conceptualization, funding acquisition.

Ethics declaration

This study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. This study was approved by the Laboratory Animal Center of Huazhong Agriculture University. (Approval No. 202603120004)

Data

The nucleotide sequence of Staphylococcus phage SAP_2 reported herein has been submitted to GenBase with assigned accession number: C_AA130092.

Data availability

Data will be made available on request.

Funding sources

This study was supported by the National Natural Science Foundation of China grant (U23A20241), the National Key Research & Development Program grant (2022YFA1304104), the earmarked fund for CARS-41, and the Fundamental Research Funds for the Central Universities grant (2662025PY032).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We sincerely thank Yingying Xu of Huazhong Agricultural University for providing instructions on electron microscopy observation.

Handling Editor: Dr. Siyun Wang

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.crfs.2026.101569.

Contributor Information

Pan Tao, Email: taopan@mail.hzau.edu.cn.

Haihong Hao, Email: haihong_hao@aliyun.com, haohaihong@aliyun.com.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

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Supplementary Materials

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Data Availability Statement

Data will be made available on request.

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