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. 2026 Feb 26;110(1):82. doi: 10.1007/s00253-026-13708-1

Characterization, genomic analysis, and decontamination application of a novel Vibrio phage BUCT787 on salmon fillets

Jiteng Xiao 1, Haoyue Dai 2, Xiaoqian Lv 3, Meng Li 2, Mengzhe Li 1,✉, Yigang Tong 1,✉
PMCID: PMC12948841  PMID: 41748907

Abstract

Abstract

Vibrio spp. are significant zoonotic pathogens in seafood, causing human gastroenteritis and posing challenges to the aquaculture industry. The misuse of antibiotics in aquaculture has led to the rise of antibiotic-resistant strains, necessitating alternative solutions like phages for food safety and pathogen control. Herein, we isolated and characterized a novel lytic Vibrio phage, BUCT787, from salmon aquaculture environments. Genomic analysis revealed that BUCT787 shared only 68% genome-wide coverage and 95.97% sequence identity with Vibrio phage 207E29.1, classifying it as a new member of an unclassified family within the Caudoviricetes order. The phage genome contained 86 predicted open reading frames (ORFs), with only 29 ORFs encoding proteins with known functions. BUCT787 exhibited stability across a wide range of temperatures (4°C–36°C) and pH conditions (4–12). To optimize infection efficiency, the optimal MOI for BUCT787 was determined to be 0.01, and the one-step growth curve revealed a latent period of 13 min, followed by a burst phase of 100 min, with a burst size of 42.1 PFU/cell. Furthermore, we developed a novel phage application platform using aerosolization technology to significantly reduce pathogen levels in closed environments and on seafood products, maintaining the freshness of salmon fillets and meeting GRAS standards. BUCT787 exhibited remarkable inhibitory activities against Vibrio spp., with an inhibition efficiency of 99.9%, and preserved the quality of salmon fillets. These findings establish BUCT787 as a promising biocontrol agent, offering a sustainable solution for enhancing seafood safety, reducing the spread of antibiotic-resistant pathogens, and supporting the sustainability of the aquaculture industry.

Key points

• Isolated a novel lytic Vibrio phage BUCT787 with 68% coverage and 95.97% identity.

• Phage BUCT787 showed efficient lytic activity and a wide pH and temperature tolerance range.

• Developed a phage aerosolization platform that reduced Vibrio contamination by 99.9% on salmon fillets.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-026-13708-1.

Keywords: Bacteriophage, Biocontrol, Seafood, Vibrio, Antibiotic alternatives

Introduction

Food safety remains a critical global health issue, with foodborne illnesses imposing substantial economic burdens and health risks (Powell 1999). According to the World Health Organization, an estimated 600 million people worldwide suffer from foodborne diseases each year, leading to 420,000 deaths and a loss of 33 million disability–adjusted life years (DALYs) (https://www.who.int/news-room/fact-sheets/detail/food-safety). Among various foodborne pathogens, Vibrio spp. are particularly concerning due to their widespread prevalence in marine environments, posing a significant threat to seafood safety (Joseph et al. 1982). These pathogens frequently caused disease outbreaks associated with seafood, such as shrimp, salmon, and shellfish, contributing to serious public health concerns and economic losses in the aquaculture industry (Lee et al. 2023).

Traditional methods for controlling Vibrio spp. contamination, such as heat treatment and high-pressure processing, effectively inactivate Vibrio spp. in seafood. However, these techniques can also cause protein denaturation, potentially resulting in undesirable flavors and odors (Sucena Afonso et al. 2024; Wang et al. 2015). As consumer preferences shift towards raw or lightly cooked seafood, there is a growing need for anti-bacterial methods that meet this demand while ensuring safety. Furthermore, the misuse of antibiotics has led to the emergence of antibiotic-resistant Vibrio strains, further complicating control efforts (D’Costa et al. 2011). The U.S. FDA’s guidance for fish and fishery products, Hazards and Controls Guidance (Chapter 11), emphasizes minimizing the use of synthetic antimicrobial agents in aquaculture to reduce reliance on therapeutic drugs such as antibiotics and other chemical compounds, driving the pursuit of innovative and sustainable alternatives.

Bacteriophages (phages) are viruses that specifically infect bacteria, offering a promising alternative for bacterial control due to their high specificity, safety, and abundance in the natural environment (Gordillo Altamirano and Barr 2019). The application of phages in food safety is gaining attention, particularly in targeting problematic pathogens like Vibrio spp. Phages can selectively reduce bacterial populations without affecting beneficial microbiota or altering the sensory qualities of food (Chang et al. 2022; Wang et al. 2024). Numerous studies have investigated the use of phages in food biocontrol, including applications in food preservation, safety, and microbial detection (Endersen and Coffey 2020). Several phage preparations have received FDA approval for bacterial decontamination. In 2006, the U.S. FDA approved the phage cocktail Listex_P100 for controlling Listeria in food (GRN No. 198). In 2013, the FDA recognized the phage formulation SalmoFresh™ targeting Salmonella as Generally Regarded As Safe (GRAS) and approved it for marketing (GRN No. 435). In 2014, the FDA concluded that the phage cocktail ListShield™ was safe for use as an antibacterial preparation targeting Listeria monocytogenes in food (GRN No. 528). Subsequently, the FDA also approved phage cocktails PhageGuard E™ for biocontrol of E.coli (GRN NO. 834). Currently, no phage-based products targeting Vibrio spp. have been approved by the FDA for use in fish or other aquatic products. Nevertheless, several studies on Vibrio-infecting phages, including VPK8, have shown effective inhibition of Vibrio harveyi and other pathogenic species, leading to reduced disease incidence in mariculture (Jintasakul et al. 2025). These findings highlight the potential of Vibrio phages for biological disease control. However, the number of isolated Vibrio phages remains insufficient to fully address pathogenic threats in aquaculture, underscoring the urgent need to identify more phages with strong lytic activity. Therefore, isolating and characterizing Vibrio-associated phages is crucial for advancing the aquaculture industry and ensuring the hygiene and safety of aquatic food products.

In this study, we identified a novel strain of Vibrio spp. from salmon aquaculture pond water and isolated a new virulent Vibrio phage designated as BUCT787 with the newly-identified strain as the host. We conducted a comprehensive analysis of its physiological properties and genomic characteristics. Additionally, we assessed the inhibitory effects of phage on host bacterial growth in both liquid culture and artificial Vibrio-spiked salmon. Our results demonstrate that BUCT787 holds significant potential for biocontrol applications in Vibrio spp.’ decontamination in aquatic products. Furthermore, we developed a novel antibacterial platform and evaluated its efficacy, providing a new phage-based solution to control Vibrio spp. in food safety and aquatic products.

Materials and methods

Bacterial isolation and identification

Water samples were collected from salmon aquaculture ponds in Qingdao, Shandong Province, China. To maximize the diversity of isolated Vibrio species, the 50 mL sample was centrifuged at 7000 rpm for 15 min to obtain the precipitate, which was then resuspended in 100 µL of phosphate buffer saline (PBS, Biosharp, China) (Nnadozie and Ngoni 2023). The resuspended sediments were plated onto Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS agar, Hopebio, China), and incubated at 30°C for 12 h. Blue-green and yellow colonies were selected using a sterile inoculation loop and then purified by streaking onto Zobell Marine Agar 2216 (2216E agar, Hopebio, China). The Vibrio isolates were purified through three rounds of streaking. Subsequently, the strains were confirmed by amplification of the 16S rRNA genes using PCR with the primers 27 F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (5′-CGG TTA CCT TGT TAC GAC TT-3′) (Weisburg et al. 1991). Cultures were incubated overnight in Zobell Marine Liquid 2216 (2216E liquid, Hopebio) at 30°C with shaking at 200 rpm, and stored in 20% (v/v) glycerol solution at −80°C.

Phage isolation, purification, and genomic analysis

We used the newly identified Vibrio spp. as the host to isolate phages from the same salmon pond water. Water samples were centrifuged at 8000 rpm, and then the supernatant was filtered through a 0.22 μm filter to remove bacterial contaminants. 200 μL of the filtrate was incubated with bacterial cultures to enrich potential phages. The enriched samples were then centrifuged and filtered to obtain phage-containing solutions, which were detected using the double-layer agar method. Phage purification was conducted by picking up single plaques and dissolving them in PBS, followed by repeated centrifugation, filtration, and forming plaques with the double-layer agar method at least three times. High titer phage stocks (109 PFU/mL) were stored at 4°C and −80°C in a 20% (v/v) glycerol solution.

Vibrio cyclitrophicus (V. cyclitrophicus) BUCT5157 and its lytic phage BUCT787 were isolated and preserved in the Laboratory of Environmental Microbiology, Beijing University of Chemical Technology. Both the bacterial strain and phage are available from the corresponding author upon reasonable request for the purpose of replicating the experiments described in this study.

Phage genomic DNA extraction, sequencing, and bioinformatic analysis

Phages were concentrated using a 30% sucrose density gradient ultracentrifugation, followed by RNase-A and DNase-I treatments to eliminate bacterial RNA and DNA. Phage genomic DNA was extracted using the phenol–chloroform method (NE0210, Leagene). Sequencing was performed on the Illumina NovaXplus platform, and sequencing data quality was evaluated with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Trimmomatic-0.36 was used to remove adapter sequences and low-quality reads (Bolger et al. 2014). SPAdes-3.13.0 was used for clean reads assembly (Bankevich et al. 2012). The phage genome sequence was compared to viral sequences in NCBI databases with the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990). Open reading frames (ORFs) and functional genes were predicted using the online Rapid Annotation using Subsystem Technology (RAST) server and Prokka, followed by validation through the online InterPro tool and BLAST analysis (Seemann 2014; Aziz et al. 2008; Waterhouse et al. 2018; Blum et al. 2025).

Transmission electron microscopy

Phages were concentrated by centrifugation at 35,000 × g at 4°C using a 30% (v/v) sucrose density gradient, after which the concentrated phage particles were resuspended in PBS buffer. 30 μL of phage lysate were incubated on a carbon-coated copper grid for 10 min. Excess liquid was then absorbed from the edges using filter paper, followed by a 1 min resting period. The dried support film was placed on a sealing membrane and stained with 2% (w/v) uranyl acetate for 90 s. Excess stain was absorbed with filter paper, and samples were dried for 3 h. The morphology of the phages was observed using a transmission electron microscope (JEM-1200EX, JEOL, Tokyo, Japan) at 80 kV (Bagchi 2024).

Biological characterization of the phage

Thermal and pH stability

Phage suspensions were incubated at different temperatures (− 20°C, 4°C, 16°C, 26°C, 36°C, 46°C, 56°C, and 66°C) for 120 min to determine the thermal stability. Likewise, for pH stability, the pH of the 2216E liquid medium was adjusted using 1 M hydrochloric acid (HCl) for the acidic range (pH 2–6) and 1 M sodium hydroxide (NaOH) for the alkaline range (pH 8–13). The pH values were verified with a calibrated pH meter, phages were incubated in 2216E broth at different pH levels ranging for 60 min. Phage titers were examined using the double-layer agar method, with all experiments performed in triplicate.

Optimal multiplicity of infection (MOI)

Bacterial cultures were infected with phages at varying MOI levels, including 100, 10, 1, 0.1, 0.01, and 0.001. The mixtures were incubated at 30°C for 6 h with shaking, followed by centrifugation at 8000 × g for 2 min to eliminate bacterial cells. The supernatant was then filtered through a 0.22 µm filter, and the phage titer was determined using the double-layer agar method. The MOI that yielded the highest phage titer was identified as optimal, with all conditions conducted in triplicate.

One-step growth curve

Phages were allowed to adsorb onto host bacteria in the exponential phase at the optimal MOI for 10 min. The mixture was then centrifuged at 5000 rpm for 3 min, the supernatant was discarded, and the pellets were washed three times with PBS buffer. The final resuspension was inoculated into 30 mL of 2216E broth. Starting from time zero, 1 mL aliquots were taken every 5 min for the first 20 min, followed by 10 min intervals. Each sample was filtered, and the phage titer was determined using the double-layer agar method. Three independent replicates were conducted for this experiment.

In vitro antibacterial abilities

To assess in vitro antibacterial activities, 100 µL of fresh phage lysate and 100 µL of host bacteria were added to 5 mL of 2216E broth and incubated at different MOIs. 200 µL of the samples were collected hourly, and absorbance was measured at different time points using a Multiskan FC Photometermicro (Thermo Scientific, USA). A control group without phage was included, and all experiments were conducted in triplicate.

We simulated a sterile and closed environment to evaluate the antibacterial efficacy of phage BUCT787, using a transparent plastic chamber with dimensions of 35 cm × 30 cm × 20 cm. In this enclosed environment, we placed a nebulizer containing 15 mL of phage BUCT787 at a titer of 108 PFU/mL. We placed three bacterial culture plates at different distances of 20 cm in front of the nebulizer, 20 cm on the right side, and 20 cm on the left side, and the inoculum concentration of each plate was 109 CFU/mL. The nebulizer aerosolized the phage preparation, producing particles with diameters ranging from 5 to 10 μm (CreatiPhage, China). Three bacterial culture plates, inoculated with a concentration of 109 CFU/mL, were placed within the box at different distances from the nebulizer. After spraying for 10 min, the bacterial colony counts on the surface of the culture plates were measured.

Antibacterial effects on salmon fillets surfaces

Fresh salmon fillets, purchased from a local supermarket, were prepared into thin, rectangular slices (approximately 50 g, 6 cm × 4 cm) under sterile conditions. Subsequently, these fillets were artificially spiked with V. cyclitrophicus at a final concentration of approximately 108 CFU/cm2. V. cyclitrophicus-spiked salmon fillets and a nebulizer with different treatments were respectively placed inside the simulated chamber. The control group received 5 mL of PBS. In the two treatment groups, one received a single dose of 5 mL of BUCT787 phage solution, while the other was to be taken in three doses of 5 mL each over 30 min, totaling 15 mL. Bacterial growth on the salmon surface was monitored every 30 min. Disposable swabs were used to sample 1 cm2 of the salmon surface, and the swabs were subsequently dissolved in PBS buffer. Bacterial counts were determined using TCBS agar plates. All tests were performed in triplicate.

Sensory evaluation on the freshness of salmon samples

Freshness is widely recognized as one of the most critical attributes of fish quality, playing a key role in determining the overall quality of fish and fishery products. The Quality Index Method (QIM) is a reliable and objective method widely used to determine fish freshness quality evaluation (Cheng et al. 2015). We applied a standardized sensory evaluation based on QIM to evaluate the freshness of phage-treated salmon samples. Using the QIM methodology established by Grethe Hyldig, we employed multiple weighted quality parameters and a scoring system ranging from 0 to 3 demerit points based on important sensory quality parameters of salmon (Hyldig and Green-Petersen 2005). The QIM scheme for farmed salmon included quality parameters such as color, gloss, odor, mucus, texture, and external crack, with each parameter accompanied by detailed descriptions. The QIM scheme used in this study was shown in Table 1. The scores for all the characteristics were summed to provide an overall quality index (QI), where a higher QI score indicated greater deterioration of the fish. As for the sensory evaluation, 12 specially trained panels were involved in evaluating salmon fillets with different treatments based on sensory attributes, and scores were recorded.

Table 1.

QIM scheme for salmon fillets with different treatments

Quality parameter Description Score
Color/appearance The color of fresh fish is light golden yellow, with a uniform distribution 0
The skin is less golden yellow, with a more uniform color 1
Significantly pale or discolored 2
Mucus Clear, not clotted 0
Milky, clotted 1
Yellow and clotted 2
Gloss Shiny and glossy 0
Average gloss, dullness 1
Dull, cloudy 2
Odor Fresh seaweedy, neutral 0
Weak freshness, fishy,metal 1
No fresh flavor, Sour, dishcloth 2
No fresh flavor, Rotten 3
Texture In Rigor 0
Fingermark disappears rapidly 1
Finger leaves mark over 3 s 2
Limp and inelastic 3
External crack No surface cracks 0
Few surface cracks, short and thin 1
Visible surface cracks, long and wide 2
Quality Index (0–14)
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Results

Isolation and identification of Vibrio spp. and phage BUCT787

We successfully isolated a strain of Vibrio spp. from a salmon aquaculture pond in Qingdao, Shandong, China. This strain formed yellow colonies on TCBS agar plates with a diameter of about 3 mm (Figure S1). BLAST analysis of the 16S rRNA gene sequences identified this strain as V. cyclitrophicus, a member of the Splendidus clade, and we named it as V. cyclitrophicus BUCT5157(16S rRNA SRA accession number: SRS23655124). Using V. cyclitrophicus BUCT5157 as the host, we isolated a phage from a salmonid aquaculture pond and designated it as phage BUCT787. Phage BUCT787 could form clear and regular plaques on V. cyclitrophicus BUCT5157 lawns, with an average diameter of approximately 1 mm and without visible halos (Fig. 1A). Transmission electron microscopy revealed that phage BUCT787 possessed an icosahedral head approximately 52 nm in diameter and a contractile tail about 87 nm in length (Fig. 1B).

Fig. 1.

Fig. 1

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Morphological characteristics of phage BUCT787. A Phage plaques formed on the lawn of V. cyclitrophicus BUCT5157. B Morphology of the phage observed using TEM

Genomic analysis of BUCT787

The genomic DNA of phage BUCT787 was 48,532 bp in length with a G + C content of 42.56% (Fig. 2). A comparative analysis using BLASTn revealed that phage BUCT787 shared the highest similarity with Vibrio phage 207E29.1 (GenBank: MW824374.1), showing 68% genome-wide coverage and 95.97% sequence identity. Bioinformatics analysis further confirmed that phage BUCT787 lacked genes associated with antibiotic resistance, virulence factors, and lysogeny-related elements, such as integrase, supporting its classification as a lytic phage.

Fig. 2.

Fig. 2

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Genome map of bacteriophage BUCT787 and its genetic characteristics

The genome annotation of phage BUCT787 identified 85 predicted open reading frames (ORFs), with considerable length variation ranging from 71 bp to 2,333 bp. The total length of the coding region of phage BUCT787 was 44,301 bp, which accounted for 91.28% of its genome, indicating a high gene utilization rate. This characteristic may be associated with the complexity of the phage and its environmental adaptability, and evolutionary process. A detailed list of these ORFs and their putative functions was provided in Table S1. We identified 29 ORFs that encode proteins with high homology to known proteins, while the remaining 56 ORFs were annotated as hypothetical proteins with unknown functions. Functional annotation categorized these ORFs into four modules: structure, packaging, replication, and other functional modules. The largest number of open reading frames that function as structural proteins includes tail completion protein (ORF25, ORF42), baseplate (ORF27, ORF30), head protein (ORF43, ORF45, ORF75), neck protein (ORF75), and coiled-containing protein (ORF2, ORF6, ORF28, ORF51). The tail completion protein, produced by a highly conserved gene, is thought to be located at the proximal end of the siphotail, potentially to help correct the positioning of the DNA after capsid-tail joining (Zinke et al. 2022). The neck protein acts as a symmetry adaptor between the capsid and tail, regulating the release of phage DNA into the host cell during infection. The junction between the tail and capsid exhibits a symmetry mismatch bridged by the neck, making this structural module essential for the assembly of tailed phages (Cumby et al. 2015; Greenfield et al. 2020; Aksyuk and Rossmann 2011). Packaging proteins include terminase large subunit (ORF84) and putative DNA-packaging (ORF1). Terminase large subunit explicitly recognizes phage sequences and can be precisely localized to the phage genome to initiate phage packaging (Ray et al. 2009). The replication module includes DNA helicase (ORF16, ORF19) and single-stranded DNA-binding protein (SSB, ORF56). DNA helicase is a crucial enzyme involved in nucleic acid metabolism. This enzyme uses the energy derived from nucleoside triphosphate hydrolysis to translocate along nucleic acid strands, separate the helical structure of double-stranded nucleic acid, and maintain continuous activity during DNA replication (Abdelhaleem 2010). Single-stranded DNA binding proteins are found in all areas of life and play essential roles in DNA replication, recombination, and repair by binding to single-stranded DNA with high affinity (Antony and Lohman 2019). Notably, this phage exhibits strong lysis properties, but we did not directly annotate any proteins related to cell lysis. We speculate this may be due to significant differences between this phage and others with well-annotated bacteriophage databases. Additionally, we identified three tRNA-encoding sequences in the phage genome for Arg (arginine), Ser (serine), and Gly (glycine). The presence of tRNAs in phage genomes is common and serves to compensate for the host’s tRNA deficiencies. This adaptation enhances translation efficiency and provides a competitive advantage in tRNA-depleted environments (Bailly-Bechet et al. 2007). More importantly, no lysogenic genes, mobile genetic elements, or virulence factors were identified in this phage. This indicates that the phage has a high safety profile and is suitable for use in biocontrol applications.

Comparative genomic and taxonomic analysis of BUCT787

Comparative genomics analysis between phage BUCT787 and phage 207E29.1 revealed that conserved regions are predominantly located in structural protein genes and some putative proteins (Fig. 3A). The comparisons showed low similarity between the two phages in other coding regions with differences less than 90%. To further explore the taxonomy of BUCT787, we performed a genomic comparison of phage BUCT787 with all phages sharing homology in the NCBI database. Based on the whole-genome sequence similarities calculated using tBLASTx, a proteomic tree of viral genomes was generated, revealing global genomic similarity relationships between hundreds of phages (Fig. 3B). For a more detailed phylogenetic analysis of BUCT787, we selected 27 representative phages from different families, including all Vibrio phages with similar sequences and other phages with higher similarity (Fig. 3C). According to the phylogenetic analysis, we found that phage BUCT787 is in the same evolutionary branch as its most similar Vibrio phage 207E29. It also shares a close phylogenetic relationship with Vibrio phage vB_VchM-138 (NC_019518) and Vibrio phage CP-T1 (NC_019457). They are both new members of the unclassified family in Caudoviricetes.

Fig. 3.

Fig. 3

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Phylogenetic trees formed by phage genomes. A Genome comparison of BUCT787 and Vibrio phage 207E29.1 using Easyfig 2.2.3. The arrows represented predicted open reading frames. Shading indicated nucleotide identity between sequences (71%–100%). B Phylogenetic tree using phage and all related phage genomes. Phage BUCT787 is marked with a red pentagram. C An evolutionary tree built with 27 phages with high homology to phage BUCT787. Phage BUCT787 is marked with a red pentagram.

Characterization of BUCT787

To optimize infection efficiency, we determined the optimal MOI for BUCT787 by infecting host bacterial cells with different phage titers at varying MOIs. The highest amounts of progeny phages could be produced with an MOI of 0.01, reaching approximately 2.1 × 108 PFU/mL (Fig. 4A). The one-step growth curve of BUCT787 at this MOI indicated its latent period of about 13 min, followed by a burst phase of 100 min (Fig. 4B). The burst size was calculated to be 42.1 PFU/cell. These characteristics, including a short latent period, high lytic capacity, and robust proliferation, make BUCT787 an ideal candidate for phage-based antimicrobial applications.

Fig. 4.

Fig. 4

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Biological characteristics of BUCT787. A Determination of the optimal multiplicity of infection. B One-step growth curve at MOI of 0.01. C pH stability of BUCT787 incubated at various pH for 120 min. D Thermal stability of BUCT787 incubated at various temperatures for 120 min. Data were expressed as the mean ± SD. *P < 0.05; ns, not significant

We also assessed the pH and the thermal stability of BUCT787 under various pH and temperature conditions. This phage BUCT787 exhibited high stability across a broad pH range of 4–12, retaining more than 90% of activities compared with the initial levels. Phage titers decreased under extremely acidic and alkaline conditions, with a 41% reduction in activity after 2 h of incubation at pH 3 and complete inactivation observed at pH 13 (Fig. 4C). This phage also demonstrated high thermal stability within the temperature range of 4°C to 36°C, retaining more than 90% of its activity compared to the initial level (Fig. 4D). Phage BUCT787’s activity decreased by 22% after 2 h at 46℃ and by 99% after 2 h at 66°C.

Antibacterial activities of BUCT787 in a simulated Vibrio-contaminated environment

The efficiency of viral infection is influenced by several factors, including the growth state of host cells, cell density, and susceptibility to infection (Bailly-Bechet et al. 2007). The MOI is a critical parameter, especially in the biocontrol of food microorganisms or the prophylactic phage applications (Abedon 2016). To evaluate the potential of phage BUCT787 as a biocontrol agent, we exposed V. cyclitrophicus BUCT5157 to varying concentrations of phages, with MOI ranging from 0.1 to 100. As shown in Fig. 5A, during the first 6 h, the optical density (OD) of the bacterial culture without phage BUCT787 gradually increased to 0.4, whereas the OD of the culture treated with phage BUCT787 was nearly unchanged. This indicated that phage treatment effectively suppressed the growth of V. cyclitrophicus cultures and the phage inhibition effect was most effective at an MOI of 0.1. Even after 15 h, phage treatment still demonstrated a significant inhibitory effect, as evidenced by a markedly lower OD in the phage-treated group, with the most potent inhibition observed at a MOI of 10. However, starting from the sixth hour of phage treatment, the OD of phage-treated groups gradually increased. This suggests the potential growth of phage-resistant bacterial strains under selective pressure. The appearance of phage-resistant strains is a natural outcome of the co-evolution of bacteria and phages, commonly observed in laboratory settings (Hampton et al. 2020). Then, we isolated phage-resistant mutants and tested the inhibitory activity of phage BUCT787 against both wild-type and resistant mutant strains at an MOI of 0.1. The results showed the phage exhibited almost no inhibitory activities against the resistant mutants (Fig. 5B).

Fig. 5.

Fig. 5

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In vitro antibacterial activity of BUCT787. A Antibacterial curves of phage BUCT787 to V. cyclitrophicus using different MOIs (MOI = 100, 10, 1, 0.1) in a liquid environment (2216E broth). B Antibacterial effects of BUCT787 on wild-type strains of V. cyclitrophicus. C The antibacterial effect of BUCT787 on phage-resistant mutant strains of V. cyclitrophicus. ∗∗ P < 0.01 and ∗∗∗∗ P < 0.0001. ns, not significant

To further evaluate the antibacterial effects of phage BUCT787, we evaluated its antimicrobial activities in a simulated closed environment. Current research on Vibrio phages typically utilizes direct application or coating methods in aquatic settings, seafood processing equipment, and the seafood itself (Zhang et al. 2018). In contrast, aerosol spraying provides a simpler and more uniform application method. Herein, we employed aerosolization to deploy phage BUCT787 for environmental decontamination. A phage preparation with a titer of 108 PFU/mL was aerosolized using a nebulizer, generating particles within the 5–10 μm range, with no significant loss of phage titer post-aerosolization (Fig. 6A). The nebulizer with phage BUCT787 was placed in a closed environment, and three plates inoculated with a high concentration of V. cyclitrophicus BUCT5157 were placed at different locations. After continuous nebulization for 10 min, bacterial colony counts on the plates showed a significant reduction from 108 CFU/cm2to 103 CFU/cm2, with no significant differences across the three locations (Fig. 6B). These results demonstrate that the phage BUCT787 formulation effectively inhibits bacteria on environmental surfaces. Furthermore, the similar effects observed across samples from different locations suggest that the aerosol method ensures uniform distribution, thereby enhancing its antibacterial efficacy.

Fig. 6.

Fig. 6

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Antibacterial effects of phage nebulization in a simulated Vibrio-contaminated environment. A Plaque formation of phage lysates before and after nebulization. B Phage titers before and after nebulization, using pre-nebulization phage as the control. C Anti-bacterial effects of phage aerosolized preparations on bacteria at different locations in a closed environment. Data were expressed as the mean ± SD. ∗∗∗∗ P < 0.0001, ns, not significant

Potential as a biocontrol agent in artificially Vibrio-spiked salmon fillets

Under the Chinese national standard for food safety GB 29921–2021 and the International Commission on Microbiological Specifications for Foods (ICMSF), V. parahaemolyticus levels in ready-to-eat seafood must not exceed 103 CFU/g. To evaluate the efficacy of bacteriophage BUCT787 in reducing the levels of Vibrio on food surfaces, we simulated its antimicrobial activity in a closed environment. Salmon samples were inoculated with bacterial cultures to a concentration of 10⁶ CFU/g and placed in the environment alongside a nebulizer containing phage BUCT787. We assessed the bacterial counts of Vibrio on the salmon surfaces after aerosolizing 15 mL of the phage preparation (Fig. 7A). The results showed a significant reduction in bacterial colonies in nebulizer-treated samples, with a 99% inhibition rate. Colony counts remained below 103 CFU/g within 120 min of phage treatment, meeting safety standards. However, from 60 min post-treatment, the colony counts of V. cyclitrophicus began to gradually increase, which is consistent with our previous findings and is likely attributed to the emergence of phage-resistant bacterial strains. To further enhance the antimicrobial effects of BUCT787, we increased the frequency of phage administration. Starting with the initial aerosolization, we administered aerosol treatments every 20 min, applying 15 mL of the phage BUCT787 preparation a total of three times. Following this treatment, we observed a rate of 99.9% for V. cyclitrophicus on the salmon surfaces, bringing the count well below the food safety standard of 103 CFU/g. Additionally, the emergence of drug-resistant strains was effectively delayed (Fig. 7A). We employed the Quality Indicator Method (QMI) to assess the quality of salmon. Twelve laboratory volunteers were trained, and sensory evaluation of different salmon samples was performed after passing the training. The average scores for each index indicated that the gloss, odor, surface mucus, and texture of the phage-treated samples were significantly better than those of the control group after being stored at 30℃ for 12 h (Fig. 7B). We calculated the QI index by summing the average scores of each parameter, with a lower QI indicating higher freshness. The QI results revealed that the freshness of the phage-treated samples was significantly superior to that of the control group, and multiple phage aerosol treatments further improved the freshness (Fig. 7C).

Fig. 7.

Fig. 7

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Anti-bacteria testing of phage BUCT787 on the surface of Vibrio-contaminated salmon fillets. A Bacteriostatic testing device for phage BUCT787 aerosolized formulation in a simulated closed environment. B Anti-bacterial effect of different nebulisation methods of BUCT787; number of V. cyclitrophicus colonies per square centimeter of food surface measured every 30 min. C Salmon state after application of phage BUCT787 aerosolized formulation. Data were expressed as the mean ± SD (error bars). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001

Discussion

V. cyclitrophicus often dominates disease outbreaks during the warmer spring months and has been associated with increased mussel mortalities (Li et al. 2019; Triga et al. 2023). Additionally, V. cyclitrophicus is also linked to vibriosis, particularly in European seabass and other farmed fish species during summer and autumn, with occurrences reported in spring as well (Kapetanović et al. 2022; Nuttall and Moisander 2023). However, research on phages that specifically infect V. cyclitrophicus remains limited, with only phage vB_VviC_ZQ26 infecting V. cyclitrophicus reported from coastal waters near Qingdao, China (Xiong et al. 2024). Considering the potential impact of V. cyclitrophicus on aquaculture and the quality and safety of aquatic food, it is essential to collect more phages infecting this strain for effective biocontrol. In this study, we isolated and characterized BUCT787, a novel lytic phage targeting V. cyclitrophicus. Genomic analysis revealed that BUCT787 lacks virulence and lysogeny genes, supporting its potential as a safe biocontrol agent. Phylogenetic analysis showed that it shares only 68% genome coverage with its closest known relative, phage 207E29.1, highlighting both its novelty and the broader genetic diversity of Vibrio phages, as well as the need to expand current phage genome databases.

Biological characterization showed that BUCT787 has a short latent period and high burst size, suggesting high replication efficiency and strong lytic potential. It also exhibited good stability across a wide range of pH and temperature conditions, making it well-suited for application in aquatic product processing. However, BUCT787 is sensitive to high temperatures (> 66°C), which may restrict its use in thermally processed foods. In contrast, studies have shown that E. coli phage vB_Ec_ZCEC14 can tolerate temperatures up to 80°C, and 40% of isolated Lactobacillus phages survive heating at 80°C for 5 min (Ismael et al. 2024). BUCT787 is more sensitive to high temperatures than these highly heat-resistant phages. This sensitivity may stem from significant genomic and coat protein structural differences. In addition, this may be related to the growth characteristics of its host, V. cyclitrophicus, which thrives at optimal temperatures of 25–30°C and is associated with mass mussel mortality at 27℃ (Takemura et al. 2014; Padfield et al. 2020). Temperature and pH of phage preservation are the most important factors in determining phage activities. Notably, phage BUCT787 exhibited minimal activity reduction under low-temperature and alkaline conditions, highlighting its resilience to these environmental factors. These findings underscore the potential of BUCT787's application in antimicrobial formulation development, providing a foundation for the creation of bacteriostatic agents based on this phage.

Building on these findings, we developed an aerosolized phage formulation to evaluate the potential of BUCT787 in surface decontamination applications. The results show that this disinfection method ensures uniform distribution of the phage and achieves a 99.9% reduction in bacterial counts on salmon fillets, meeting food safety standards. Aerosolized bacteriophage formulations have also shown significant potential in pathogen control across diverse environments. In hospitals, nebulized phage cocktails effectively reduce nosocomial transmission of Carbapenem-resistant Acinetobacter baumannii (CRAB), enhancing healthcare safety (Ho et al. 2016). Similarly, in the agricultural environment, continuous phage aerosol application on farm surfaces has substantially reduced Salmonella Infantis contamination (Sevilla-Navarro et al. 2024). These findings underscore the versatility of phage-based aerosols as a sustainable alternative to traditional disinfection methods.

Given the increasing demand for ready-to-eat raw seafood products, which require both microbiological safety and freshness, the application of phage BUCT787 via aerosol offers a promising approach. In addition to reducing Vibrio contamination, sensory evaluation indicated that phage treatment helped maintain product freshness, making it highly relevant for high-value perishable foods. To enhance antibacterial efficacy, we implemented a multiple-dose aerosol strategy, which both improved bacterial suppression and delayed the emergence of phage-resistant strains. However, phage-based inhibition also presents certain limitations. Although phage BUCT787 exhibits strong lytic activity, the emergence of resistant mutants after long-term exposure is a cause for concern. This aligns with previous studies showing that phage monotherapy can select for resistant strains. Yuan et al (2019) reported that the phage cocktail can significantly lower mutation frequencies and delay the emergence of resistant strains while demonstrating enhanced lytic efficacy. Consequently, future studies can focus on developing a more effective strategy by preparing phage cocktails, incorporating additional phages based on BUCT787 to address the resistance issue.

Conclusion

In summary, stringent regulation on antibiotic use in aquaculture and necessitates the development of economical and effective alternative antimicrobial agents. Although the FDA has approved several phage-based products for antibacterial use, none have been authorized for use against Vibrio spp., highlighting the need for the isolation and development of specific phages targeting this pathogen for both aquaculture and food industries. To address this gap, we isolated and characterized a novel and potent Vibrio phage, BUCT787, which exhibited robust antimicrobial activities and stable biological properties across a broad range of temperature and pH conditions. Furthermore, we developed an efficient platform for phage-based antimicrobial preparations, and simulation testing showed that phage BUCT787 achieved an antimicrobial activity of 99.9% on this platform. Additionally, the freshness of phage-treated salmon was assessed by the QIM quality index, and the results showed that the evaluation indexes such as odor and external cracks of phage-treated salmon samples were significantly better than those of the control group, indicating that phage application can effectively delay quality deterioration in seafood products. Overall, these findings lay the foundation for the development of phage cocktail preparations, contributing to the sustainable development of the aquaculture industry while protecting human health.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 115 KB) (115.1KB, docx)

Author Contributions

Jiteng Xiao wrote the main manuscript text and conducted the experiments. Haoyue Dai wrote the main manuscript text. Xiaoqian Lv and Meng Li provided research resources. Mengzhe Li and Yigang Tong contributed to the conception or design of the work. All authors reviewed the manuscript.

Funding

This research was supported by the National Key Research and Development Program of China ( No. 2025YFC3408500), National Natural Science Foundation of China (No. 82341119), and the Interdisciplinary Research Center of Beijing University of Chemical Technology (No. XK2025-05).

Data availability

All data included in this study are available upon request by contact with the corresponding author. The 16S rRNA sequence of V. cyclitrophicus BUCT5157 has been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRS23655124. The genomic sequence of phage BUCT787 has been deposited in the NCBI GenBank database with the number PQ166232.1.

Declarations

Ethical approval

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Mengzhe Li, Email: futurelmz123@163.com.

Yigang Tong, Email: tong.yigang@gmail.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 115 KB) (115.1KB, docx)

Data Availability Statement

All data included in this study are available upon request by contact with the corresponding author. The 16S rRNA sequence of V. cyclitrophicus BUCT5157 has been deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRS23655124. The genomic sequence of phage BUCT787 has been deposited in the NCBI GenBank database with the number PQ166232.1.

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