A broad-spectrum lytic bacteriophage for preventing and controlling infection of multiple vibrio in aquaculture environment

Abstract

The application discloses a broad-spectrum lytic bacteriophage for preventing and controlling infection of various Vibrio in aquaculture environment, V.alginolyticus phage vB_ValM-R37J, which was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on August 6, 2025, is located at No. 100, Hero Middle Road, Yuexiu District, Guangzhou, Guangdong, China, and has a postal code of 510070, and has a preservation number of GDMCC NO.66813-B1. The bacteriophage R37J is a candidate therapeutic bacteriophage with broad host spectrum, high lytic efficiency, genetic safety and environmental stability, and has the potential to be applied as a new biological tool for precisely preventing and controlling various Vibrio infections in aquaculture. Not only does the bacteriophage provide a new biological resource for precisely preventing and controlling aquatic pathogenic bacteria, but also provides a theoretical basis and technical support for the popularization and application of bacteriophage therapy in actual aquaculture systems.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial agents, specifically relating to a broad-spectrum lytic bacteriophage for the prevention and control of various Vibrio infections in aquaculture environments. Background Technology

[0002] With the rapid expansion of the global aquaculture industry, its importance in ensuring food security and driving economic growth is becoming increasingly prominent. According to the latest 2024 report, "The State of Fisheries and Aquaculture in the World," global fisheries and aquaculture production reached a new high, with Asian countries contributing over 90% of the output. China, as the largest aquaculture producer, saw its total aquatic product output reach 73.5759 million tons in 2024, a year-on-year increase of 3.39%. However, with the continued growth in aquaculture production, increased farming density, and rising environmental pressures, the risk of pathogenic microorganism transmission has significantly increased, seriously threatening the health of aquatic animals and the sustainable development of the industry. Among numerous pathogens, Vibrio is one of the most important pathogens causing aquatic animal diseases and economic losses globally. Vibrio is a Gram-negative bacterium widely distributed in marine environments, aquaculture environments, and animal tissues. In recent years, frequent aquatic animal disease events caused by Vibrio infection have occurred globally, drawing widespread attention. For example, in 2013, Vibrio parahaemolyticus caused acute hepatopancreatic necrosis syndrome in shrimp, leading to a massive collapse of the shrimp farming industry in Southeast Asia and Mexico; Vibrio harveyi has been reported to cause severe scale loss and muscle necrosis in Chinese hybrid grouper. To curb the spread of vibrio diseases, antibiotics have been widely used for a long time, but the resulting increase in bacterial resistance, weakened treatment efficacy, and accumulated environmental risks have become a global public health and ecological concern. In the past two decades, research has reported the identification of multidrug-resistant Vibrio strains from various sources, including Vibrio parahaemolyticus strains resistant to broad-spectrum cephalosporins, tetracyclines, and carbapenems. Against this backdrop, phage therapy, due to its high specificity and environmental friendliness, is considered an excellent alternative to environmentally friendly antibiotics and has received increasing attention.

[0003] Bacteriophages are a class of viruses that infect bacteria. They inject their genetic material into host cells by recognizing specific receptors on the bacterial surface, and then replicate and assemble using the bacterial metabolic system. Finally, they lyse the host cell to release progeny phages, thus completing the lysis of the bacteria and spreading themselves. Phage therapy utilizes this natural antibacterial mechanism, screening and applying phages with targeted bactericidal activity to treat diseases caused by bacterial infections. In aquaculture, phages can serve as an important means of precisely controlling the spread of pathogens (such as Vibrio), demonstrating good potential and application prospects for disease prevention and control. Currently, many successful cases have verified the effectiveness of phage therapy in controlling Vibrio disease in aquaculture. For example, Higuera et al. found that adding specific bacteriophages to aquaculture water increased the survival rate of Atlantic salmon infected with Vibrio anguillarum by 50%–70%, significantly improving the survival rate. Chen et al. used a mixture of bacteriophages to treat Litopenaeus vannamei infected with Vibrio alginolyticus, successfully increasing the shrimp survival rate by about 70% without observing any significant side effects such as toxicity or immunosuppression. These studies all demonstrate that phage therapy has high specificity, safety, and ecological adaptability in aquaculture, making it one of the most promising biological tools in current antibiotic alternative strategies.

[0004] Despite numerous studies demonstrating the promising potential of bacteriophages in controlling aquatic pathogens, their practical application still faces many challenges. Among these, the narrow host range is a key bottleneck restricting the widespread use of phage therapy. The range of bacteria a phage can infect constitutes its host range, which is closely related to its tail recognition structures. Phages typically recognize and bind to specific receptors on the surface of host cells through their tail structures (such as tail fimbriae or styluses), such as the lipopolysaccharide (LPS) layer, flagella, pili, teichoic acid, or outer membrane proteins of Gram-negative bacteria. They then inject themselves into the host's genome and complete the infection process, including replication, assembly, and lysis, within the cell. Most known phages exhibit strong host specificity, infecting only a limited number of species or specific strains, which limits their effectiveness in controlling a wide range of pathogens in the complex environment of aquaculture. To improve coverage, phage therapy often employs a cocktail strategy, mixing multiple phages with complementary host ranges to synergistically inhibit a broader range of pathogenic strains. However, this strategy faces certain limitations in practical applications, such as poor component compatibility and stability, complex interactions, and high research and development costs. Against this backdrop, isolating and screening virulent bacteriophages with a broad host range is of great significance. On the one hand, broad-spectrum bacteriophages can recognize and lyse multiple pathogenic strains, helping to improve the control effect against infections caused by diverse pathogens; on the other hand, their broad-spectrum characteristics are expected to simplify formulation, reduce production costs, and improve the feasibility and economics of practical application. Summary of the Invention

[0005] The purpose of this invention is to provide a broad-spectrum lytic bacteriophage for the prevention and control of various Vibrio infections in aquaculture environments and its application.

[0006] The first objective of this invention is to provide bacteriophage V. alginolyticus phage vB_ValM-R37J, which was deposited on August 6, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO.66813-B1.

[0007] The second objective of this invention is to provide a microbial preparation for the prevention and control of various Vibrio infections in aquaculture environments, which contains the aforementioned bacteriophage V. alginolyticus phage vB_ValM-R37J as an active ingredient.

[0008] A third object of the present invention is to provide the use of the above-mentioned bacteriophage V. alginolyticus phage vB_ValM-R37J or microbial preparations in the prevention and / or killing of Vibrio.

[0009] Preferably, the Vibrio species are V. alginolyticus, V. parahaemolyticus, V. owensii, V. campbellii, V. harveyi, V. splendidus, V. tubiashii, V. metschnikovii and / or V. brasiliensis.

[0010] A fourth object of the present invention is to provide the use of the above-mentioned bacteriophage V. alginolyticus phage vB_ValM-R37J or microbial preparation in the preparation of products for controlling Vibrio infection in aquaculture.

[0011] Preferably, the product includes pharmaceuticals, disinfectants, or water purifiers.

[0012] Preferably, the Vibrio infections include infections with V. alginolyticus, V. parahaemolyticus, V.owensii, V. campbellii, V. harveyi, V. splendidus, V. tubiashii, V. metschnikovii, and / or V. brasiliensis.

[0013] A fifth objective of this invention is to provide a product for controlling Vibrio infection in aquaculture, which contains the aforementioned bacteriophage Vibrio alginolyticus phage vB_ValM-R37J or a microbial preparation.

[0014] A sixth objective of this invention is to provide a method for in vitro prevention and control of harmful Vibrio infections. This method utilizes the lytic action of the aforementioned bacteriophage *Vibrio alginolyticus* phage vB_ValM-R37J or a microbial preparation to kill Vibrio in vitro. Further, the Vibrio species are *V. alginolyticus*, *V. parahaemolyticus*, *V. owensii*, *V. campbellii*, *V. harveyi*, *V. splendidus*, *V. tubiashii*, *V. metschnikovii*, and / or *V. brasiliensis*.

[0015] The inventors isolated a novel Vibrio phage, vB_ValM_R37J (R37J for short), from wastewater from the Xiamen seafood market. Using *V. alginolyticus* D61_T3_15_W4, a pathogen isolated from aquaculture environments, as a host, they systematically evaluated its biological characteristics and therapeutic potential. The results showed that R37J belongs to the myocylial phage family and possesses rapid adsorption (93.7% adsorption rate in 5 minutes), a moderate latency period (approximately 30 minutes), and a high lysis rate (138.5 ± 16.3 PFU cells). -¹) exhibits typical "highly efficient lytic" infection characteristics, making it suitable for rapid antibacterial activity and therapeutic scale-up. Its genome does not carry antibiotic resistance, virulence factors, or lysogen-related genes, demonstrating good genetic safety. It also contains multiple accessory metabolic genes, potentially enhancing its ability to regulate host metabolism during infection. Furthermore, R37J encodes multiple tail fiber structural proteins, endowing it with broad host lytic activity, effectively infecting 41 strains belonging to 8 Vibrio species (51.9% of the total tested strains), with an infection rate as high as 61.5% against aquaculture-related strains. Simultaneously, R37J maintains high activity across a wide environmental range of 4°C–37°C and pH 6–9, making it suitable for typical aquaculture water conditions. Finally, bactericidal experiments verified its rapid, efficient, and sustained antibacterial ability, exhibiting stable lytic activity (>62%) under various MOI conditions. In summary, phage R37J is a candidate therapeutic phage with a broad host spectrum, high lysis efficiency, genetic safety, and environmental stability, possessing the potential to be widely applied in aquaculture as a novel biological tool for the precise control of various Vibrio infections. This not only provides new biological resources for the precise control of aquatic pathogens but also offers theoretical basis and technical support for the widespread application of phage therapy in actual aquaculture systems.

[0016] Preservation Instructions

[0017] Vibrio alginolyticus phage vB_ValM-R37J was deposited on August 6, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC NO.66813-B1. Attached Figure Description

[0018] Figure 1 Morphage R37J morphology and infection kinetics. a: Morphological characteristics of phage R37J plaques 3, 8, and 24 hours after infection. Scale bar in single plaque images is 250 μm; b: Transmission electron microscopy image of phage R37J. Scale bar is 200 nm; c: Adsorption efficiency of phage R37J. Results showed that the adsorption rate exceeded 90% within 5 min of infection onset, and this time point was selected as the adsorption time in the one-step growth curve experiment; d: One-step growth curve of phage R37J. In the one-step growth curve experiment, the time axis in the figure includes the initial adsorption and centrifugation to remove unadsorbed phages. The formal culture start time was 0 min, used for subsequent analysis of infection kinetic parameters such as latency, lysis period, and outbreak size. Each data point represents the mean ± standard error of three biological replicates.

[0019] Figure 2. Circular genome map of bacteriophage R37J. The outermost circle shows the location and transcription direction of each ORF in the bacteriophage R37J genome, with different colors representing the predicted functional category of the encoded protein; the innermost circle shows the GC content distribution.

[0020] Figure 3 Classification of bacteriophage R37J. a: Circular phylogenetic tree generated by VIPTree; b: Phylogenetic tree containing R37J and other closely related bacteriophages constructed using the Virus Classification and Tree Construction Online Service (VICTOR); c: Heatmap generated by the Viral Genome Distance Calculator (VIRIDIC), combining genome similarity values ​​(right half) and alignment metrics (left half and top annotations).

[0021] Figure 4 The host range and infectivity of bacteriophage R37J. Different colors in the figure represent the species of each host bacterium, and different shapes (circles and rhombuses) represent the origin of the host and its habitat type. Hosts that can be lysed by bacteriophage R37J are marked with stars, and the differences in the resulting lysis plaques are distinguished by different colors.

[0022] Figure 5 Stability assessment of bacteriophage R37J under different environmental conditions. a: Stability of bacteriophage R37J at five temperature conditions (4°C, 25°C, 37°C, 45°C, 55°C); b: Stability of bacteriophage R37J at different pH conditions (pH 2–12). Data are the average of three replicates. Different letters in the figure indicate significant differences (P<0.05).

[0023] Figure 6 The bactericidal effect of bacteriophage R37J. a: Bactericidal curves of bacteriophage R37J under different MOIs (0.01, 0.1, 1, 10, 100). Each data point represents the mean ± standard error of 8 biological replicates; b: Bactericidal efficiency of bacteriophage R37J at 3, 8, and 24 h post-infection. Data are the mean of 8 replicates, and different letters in the figure indicate significant differences (P < 0.05). Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] Example 1:

[0026] 1. Materials and Methods

[0027] 1.1 Phage Isolation and Purification

[0028] This study used *V. alginolyticus* D61_T3_15_W4 as the host for phage isolation. This *V. alginolyticus* was isolated from the marine environment of a shrimp farm in Xiaoyangjia Village, Ningbo City, Zhejiang Province. Its 16S rRNA gene sequence showed 99.79% similarity to the type strain *V. alginolyticus* NBRC 15630. The host was cultured in nutrient-rich 2216E liquid medium (10 M peptone, 2 M yeast extract, artificial seawater, pH 7.5) at 28℃ and 150 rpm. -1 Dark incubation was performed under controlled conditions. Wastewater collected from the Maojie Seafood Market (Xiamen, China) was used as the water sample for phage screening. The water sample was filtered through a 0.22 μm membrane (Millipore, MA, USA) to remove large particles and bacteria, and stored at 4°C until use. 500 μL of the filtered, bacteria-free water sample was added to a Vibrio bacterial culture in the exponential growth phase and incubated in a shaker at 28°C for 24 h to enrich potential phages. After incubation, the enriched solution was filtered through a 0.22 μm pore size filter to obtain the final enriched virus solution after removing bacteria. Subsequently, approximately 5 μL of the enriched virus solution was titrated onto a double-layer plate prepared from a mixture of 2216E medium and bacteria, and incubated overnight at 28°C. If plaques are found, individual plaques are collected and stored in SM buffer (100 mM NaCl, 8 mM Tris-HCl, 50 mM MgSO4·7H2O, MilliQ water). The phages are then purified using the bilayer plate method, and at least five rounds of purification are performed to obtain pure phages with consistent morphological characteristics (Chen et al., 2023).

[0029] 2.2 Phage Amplification and Enrichment

[0030] To obtain sufficient phage for subsequent transmission electron microscopy observation and nucleic acid extraction experiments, purified phage was added to a certain volume of Vibrio alginolyticus D61_T3_15_W4 for liquid co-culture, gradually scaling up to a 1 L culture system. DNase and RNase were added to digest the free nucleic acids in the culture medium, and NaCl was added to promote phage detachment from Vibrio fragments. Subsequently, the liquid was centrifuged (8,000 × g, 4 °C, 15 min) and 0.22 μm was used to remove cell debris. A final concentration of 100 g / L was added to the filtrate. -1The phage was precipitated overnight at 4°C using PEG 8000 and centrifuged again (8,000×g, 4°C, 60 min) to collect the phage pellet. The collected phage pellet was washed with chloroform to remove impurities and then subjected to CsCl density gradient ultracentrifugation (140,000×g, 4°C, 4 h) to collect viral bands. The collected viral bands were centrifuged using a 30 kDa ultrafiltration tube (5,000×g, 4°C, 5 min) to remove excess cesium chloride solution, and repeatedly washed with SM buffer before being stored at 4°C.

[0031] 2.3 Phage Chloroform Sensitivity Test

[0032] To detect whether the outer capsid of bacteriophages contains lipids and whether there is a lipid membrane surrounding the capsid, 0%, 2%, 20%, and 40% of the volume of chloroform reagent were added to bacteriophage solutions containing the same concentration, respectively. After mixing, the solutions were incubated in the dark for 30 min (Chen et al., 2023). After centrifugation (5,000×g, 4℃, 5 min), the supernatant bacteriophage sample was collected, and the morphology and number of phage plaques were examined using the double-layer plate method to determine whether there were significant changes in the morphology and number of phage plaques after chloroform treatment.

[0033] 2.4 Transmission electron microscopy observation

[0034] The morphological characteristics of bacteriophages were observed using transmission electron microscopy (TEM) (Yang et al., 2017). First, the carbon support membrane was hydrophilically treated using a glow discharge apparatus to facilitate sample adsorption onto the membrane. Then, 3 μL of dialyzed bacteriophage solution was dropped onto the carbon support membrane and allowed to stand for 3 min to adsorb. The sample was then removed from the carbon support membrane using filter paper. Two 5 μL aliquots of 2% (w / v) phosphotungstic acid were prepared. Once no obvious watermarks were visible on the carbon support membrane, the first aliquot was added for negative staining and immediately blotted dry. The second aliquot was then added for 1-2 min, blotted dry with filter paper, and air-dried until completely dry. Observation was performed using a JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan) at 80.0 kV, and images were acquired using a digital charge-coupled device (DC-CCP) camera.

[0035] 2.5 Determination of phage adsorption rate and one-step growth curve

[0036] To determine the adsorption dynamics of bacteriophages, the host bacteria were cultured to the exponential growth phase (~10⁻¹⁰). 7 cell mL -1After inoculation with a certain amount of phage, the multiplicity of infection (MOI) was adjusted to approximately 0.01–0.03. The mixture was incubated at 28°C and 150 rpm for [time missing]. -1 Under dark conditions, samples were collected at 0, 5, 10, and 15 min and centrifuged (12,000 × g, 28 °C, 2 min). The supernatant was collected, and the number of unadsorbed phages was determined using the double-layer plate method. Three parallel samples were set up at each time point to obtain replicate data. In this study, the one-step growth curve of phages was determined according to the method of Cai et al. (Cai et al., 2023). After determining the appropriate adsorption time based on the adsorption kinetics results, the host bacteria and phages in the exponential growth phase were thoroughly mixed at room temperature and adsorbed in the dark. The mixture was centrifuged (8,000 × g, 4 °C, 5 min), and the supernatant was discarded to remove unadsorbed phages. This step was repeated twice. Subsequently, the resulting host precipitate was resuspended in 2216E liquid medium and centrifuged at 28 °C, 150 rpm for 2 min. -1 The phages were cultured in the dark. Samples were taken every 10 minutes during the culture process, and the infectivity of the phages was determined using the double-layer plate method. The culture time was approximately 90 minutes. Three parallel control groups were set up to obtain replicate data.

[0037] 2.6 Determination of the host range of bacteriophages

[0038] After culturing the host bacteria to the logarithmic growth phase in 2216E liquid medium, 1 mL of bacterial suspension was mixed with 5 mL of 0.5% 2216E agar medium and immediately poured into a pre-prepared 2216E solid plate to prepare a bacterial double-layer plate. Subsequently, 5 μL of high abundance (~10⁻⁶) bacterial culture was dropped onto the surface of the double-layer plate. 10 PFU mL -1 Phage suspensions were prepared, with SM buffer used as a negative control. Plaque formation was observed after incubation at 28°C (Yang et al., 2025). The lytic ability of host bacteria susceptible to phage infection was assessed using a dilution titration method. For each susceptible host bacterium, a phage gradient dilution (abundance 10-1) was used. 10 10 9 10 8 10 7 10 6 10 5 10 4 10 3 PFU mL -1 Titrate bacterial double-layer plates and record the differences in lysis plaque formation.

[0039] 2.7 Environmental stability of bacteriophages

[0040] To assess the environmental stability of bacteriophages, the double-layer plate method was used to detect changes in phage infectivity over time under different temperatures and pH conditions (Yang et al., 2025). In the thermal stability assay, phages with the same abundance (approximately 10⁻⁶) were plated together. 7 PFUmL -1 Phage suspensions were aliquoted into 10 mL centrifuge tubes and incubated at 4, 25, 37, 45, and 55 °C, respectively. Phage infectivity was assessed at 0, 3, 24, and 48 h. For pH stability testing, the pH of the SM buffer was adjusted to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 using a precision pH meter. Subsequently, phage suspensions of the same abundance (10⁻⁶) were incubated at 10 °C and 12. 7 PFU mL -1 Phage fluid was aliquoted into 10 mL of SM buffer at different pH values, and samples were taken at 0, 3, 24, and 48 h. Changes in infectivity were detected using the double-layer plate method. The survival rate of phages under different conditions was calculated by comparing the infectivity at each time point with the initial infectivity to assess their stability. Three parallel controls were set up for each treatment in the experiment.

[0041] 2.8 Determination of phage lysis activity

[0042] To evaluate the lytic activity of bacteriophages against host bacteria under different MOI conditions, the OD of the host bacteria was monitored in real time using a multi-functional microplate reader. 600 The optical density (OD) was measured at 600 nm (Chen et al., 2023). Specifically, fresh host bacterial culture in the logarithmic growth phase was inoculated into 96-well plates, and a certain amount of bacteriophage was added to achieve MOIs of 0.01, 0.1, 1, 10, and 100, respectively. A control group was set up with only host bacterial culture. The 96-well plates were placed in a multi-functional microplate reader and cultured at 28°C in the dark. The OD of the host bacteria was measured every 30 min. 600 The values ​​were continuously monitored for 24 hours. Eight parallel replicates were set up for each treatment group to obtain reliable data.

[0043] 2.9 Nucleic acid extraction from bacteriophages

[0044] This study used the phenol-chloroform extraction method to extract nucleic acids from bacteriophages (Yang et al., 2025). First, 10 μL of proteinase K solution (20 mg / mL) was added sequentially to the high-abundance bacteriophage solution. -1100 μL of SDS solution (10% wt / vol) and 10 μL of EDTA solution (0.5 M, pH 8.0) were added to the sample. After thorough mixing, the sample was digested in a 55°C metal bath for 3 h. After digestion, an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) mixture was added to the sample, and the mixture was thoroughly mixed. The sample was then centrifuged at 12,000×g for 10 min at 4°C, and the supernatant was collected. This step was repeated twice to completely remove the protein. Next, an equal volume of chloroform-isoamyl alcohol (24:1) mixture was added to the collected supernatant, and the sample was centrifuged for 10 min (12,000×g, 4°C). The supernatant was collected and mixed with an equal volume of isopropanol. After mixing, the sample was incubated at -20°C overnight to precipitate the DNA. Subsequently, the sample was centrifuged at 12,000×g for 10 min at 4°C, the supernatant was discarded, and the nucleic acid precipitate was washed with pre-cooled 70% ethanol solution and centrifuged for 5 min under the same conditions. This step was repeated twice to remove residual impurities. Finally, the supernatant was discarded, and after the DNA precipitate was allowed to air dry, the DNA was dissolved in TE buffer (10 mmol Tris-HCl, 1 mol EDTA, pH 8.0) and stored at -20°C for later use.

[0045] 2.10 Phage Genome Sequencing and Analysis

[0046] The phage genome sequencing and assembly were completed by Shanghai Hanyu Biotechnology Co., Ltd. (China). The sequencing library was provided by NEBNext. ® Ultra TMDNA library preparation kits were constructed, and sequencing was performed using the Illumina HiSeq 4000 platform. Trimmomatic v0.32 and Velvet v1.2.03 software were used for quality control and genome assembly of the raw data (Zerbino et al., 2008; Bolger et al., 2014). Phage gene sequences were adjusted for start positions using Phageterm software to analyze phage ends and packaging mechanisms (Garneau et al., 2017). The online software PhageAI (Tynecki et al., 2020) was used to analyze phage lysis probability. The online server GeneMarkS was used to identify putative open reading frames (ORFs) of phages. The functionality of ORFs was annotated by comparing the online BLASTP database with the non-redundant (nr) database of the National Center for Biotechnology Information (NCBI) (Altschul et al., 1997). To assess whether bacteriophages carry potential risk genes, virulence and resistance genes in the bacteriophage genome were detected using the Virulence Factor Database (VFDB) and the Comprehensive Antibiotic Resistance Database (CARD) (Liu et al., 2022; Alcock et al., 2020). Furthermore, tRNA-coding sequences in the bacteriophage genome were identified using tRNAscan-SE v.2.0 (Chan et al., 2021). A visualization of the functional modules and distribution of the genome was created using the Proksee tool (Grant et al., 2023). To further elucidate the spatial conformation of key structural proteins in bacteriophages, the online resource AlphaFold (Varadi et al., 2022) was used to predict the three-dimensional structure of bacteriophages, focusing on tail proteins and receptor-binding proteins closely related to the morphology, infection mechanism, and host binding ability of bacteriophages.

[0047] 2.11 Phage phylogenetic and taxonomic analysis

[0048] The complete genome sequences of bacteriophages were submitted to NCBI for BLASTN alignment to retrieve similar viral genome sequences. A whole-genome sequence similarity analysis of the bacteriophage genomes was then performed using the ViPTree (Virus Proteomic Tree Server) platform. Based on the similarity, a bacteriophage similarity network was constructed to provide a preliminary understanding of the phylogenetic relationships and possible taxonomic positions among bacteriophages (Nishimura et al., 2017). To further refine the phylogenetic relationships among bacteriophages, similar viral sequences obtained from the NCBI BLASTN alignment and those identified in the ViPTree analysis were jointly submitted to the VICTOR platform (Virus Classification and Tree Building Online Resource). A phylogenetic tree was constructed using the Genome-BLAST Distance Phylogeny (GBDP) method (Meier-Kolthoff et al., 2022). This method infers branch support rates from 100 pseudo-guided replications and visualizes the tree after rooting it at the midpoint, clearly demonstrating the phylogenetic relationships between bacteriophages and related viruses. To quantitatively assess the similarity between bacteriophage genomes, the VIRIDIC (Virus Intergenomic Distance Calculator) tool was used to calculate the percentage of similarity and distance matrix between bacteriophage genomes based on pairwise intergenomic comparisons (Moraru et al., 2020). Based on the species and genus classification criteria established by the International Committee on Taxonomy of Viruses (ICTV), combined with similarity thresholds, quantitative evidence was provided for the classification and identification of bacteriophages (Zhu et al., 2022).

[0049] 2 Results

[0050] 2.1 Morphological characteristics of bacteriophage R37J

[0051] This study successfully isolated a bacteriophage from wastewater in the Xiamen seafood market, Fujian Province (118.10°E, 24.45°N) using *Vibrio alginolyticus* D61_T3_15_W4 as the host, and named it vB_ValM_R37J (hereinafter referred to as R37J). Plaque morphology observation showed that R37J rapidly expanded on double-layer plates. After 3 hours of incubation, the plaques formed on the bacterial flora were approximately 0.19 ± 0.02 mm in diameter, exhibiting a relatively clear central area and a blurred and irregular edge. Figure 1 a) After 8 h of incubation, the diameter of the plaques increased to 0.51 ± 0.02 mm, and the central transparent area significantly expanded. During this period, the average growth rate of the plaque diameter was 0.06 mm / h. -1 Subsequently, the plaque expansion rate slowed significantly, with the plaque diameter increasing to only 0.57±0.01 mm after 24 h of culture. Transmission electron microscopy revealed that R37J possessed a retractable tail (approximately 123.41±2.07 nm in length) and distinct tail filaments, consistent with typical characteristics of myotail phages, while its head exhibited an icosahedral shape (approximately 108.63±1.04 nm in length and 98.49±0.47 nm in width). Figure 1 b). The chloroform sensitivity test results showed that the morphology and number of plaques of R37J did not change significantly after chloroform treatment, indicating that its structure does not contain a lipid envelope that is easily damaged by organic solvents.

[0052] 3.2 Infection kinetics of bacteriophage R37J

[0053] Adsorption experiments of bacteriophage R37J with the host bacterium V. alginolyticus D61_T3_15_W4 showed that the adsorption rate of bacteriophage R37J reached 93.7% after 5 min of contact with the host bacterium, and nearly 99.5% after 10 min. Afterward, the adsorption rate did not change significantly, indicating that most of the bacteriophage had completed the adsorption process within the first 10 min, and the adsorption rate tended to stabilize. Figure 1 c). This result indicates that phage R37J can rapidly recognize and attach to the surface of host cells within a short period of time, demonstrating highly efficient infection initiation capabilities. One-step growth curve experiments showed that the number of phages did not increase significantly within 30 minutes after infection, indicating that this stage is the latent period, during which the phage completes steps such as genome injection, replication, and assembly. Figure 1 d). Thirty minutes after infection, the number of bacteriophages began to increase rapidly, entering the lysis phase, and reached a stable level after 50 minutes. Based on the experimental results, the lysis rate of R37J was 138.5 ± 16.3 PFU cells. -1This indicates that it can release a large number of mature progeny phages within a single infection cycle. Infection kinetics show that phage R37J can rapidly adsorb onto host cells and achieve a high lysis rate after a relatively short incubation period, demonstrating its potential for efficient amplification and spread within the host bacterial population.

[0054] 3.3 Genomic characteristics of bacteriophage R37J

[0055] The genome of bacteriophage R37J is linear double-stranded DNA (dsDNA), 155,468 bp in length, with a GC content of 40.15%. A total of 217 open reading frames (ORFs) were predicted across the genome, of which 135 (62.21%) encode hypothesized proteins, and the remaining 82 ORFs, after functional annotation, were identified as being closely related to key biological processes in the bacteriophage. Figure 2Specifically, 30 ORFs are related to nucleic acid synthesis, replication, and repair processes, encoding core enzymes such as DNA polymerase, DNA primase, DNA ligase, and endonuclease subunit; 34 ORFs participate in phage structure construction and assembly, encoding key structural proteins such as large / small terminase protein, portal protein, baseplate wedge protein, tail tube protein, tail fiber protein, neck protein, and major capsid protein; three cleavage-related genes, holin, peptidase M15, and soluble lyticmurein transglycosylase, were identified in the genome; and a total of 10 auxiliary metabolic genes (AMGs) were identified, namely anaerobic ribonucleoside-triphosphate reductase (NrdD), anaerobic ribonucleoside-triphosphate reductase-activating protein (NrdG), asparagine synthase-related protein, thymidylate synthase (TS), dihydrofolate reductase (DHFR), and peptide. The bacteriophage contained deformylase (PDF), 3-deoxy-7-phosphoheptulonate (DAHP) synthase, chorismate mutase, cobalamin synthase (CobS), and dUTPase. Furthermore, no tRNA gene was detected in the R37J phage genome, nor were any functional elements such as integrases related to virulence factors, antibiotic resistance, or lysogeny found, suggesting high biocompatibility for its application.

[0056] 3.4 Classification of bacteriophage R37J

[0057] BLASTN alignment of the phage R37J genome sequence in the NCBI database showed extremely low similarity to known sequences. The top match was a bacterial genome, *Symbiopectobacterium purcellii* (Querycover=0.0%, identity=76.61%), followed by *Acinetobacter phage vB_AbaM_DLP3* (Querycover=0.0%, identity=84.71%) and *Acinetobacter phage vB_AbaM_DLP1* (Query cover=0.0%, identity=84.71%). This result indicates that phage R37J has extremely low similarity to known phages in the NCBI database, suggesting a high degree of genomic uniqueness. A circular phylogenetic tree of the whole phage genome constructed using VIPTree showed that phage R37J was divided into a separate new branch, and this independent evolutionary branch also suggests high genetic uniqueness. Figure 3 a). In the phylogenetic tree, the closest phylogenetic neighbor is the Ackermannviridae family, whose bacteriophages typically host Gram-negative bacteria, especially the Enterobacteriaceae and Vibrionaceae families. Further phylogenetic tree construction using VICTOR analysis, and following the ICTV-recommended criteria for new species (genome similarity <95%) and new genera (similarity <70%), showed that bacteriophage R37J is the closest phylogenetically neighboring marine viruses AG_341_P01 and AG_345_O18 with unknown host classifications. However, R37J still forms a separate branch and cannot be assigned to any existing genus. Figure 3 (b) (Moraru et al., 2020; Turner et al., 2021). Meanwhile, VIRIDIC analysis showed that the highest similarity score between phage R37J and other phage genomes was only 1.0, and most of the compared genomes had similarity scores of 0.1 or even 0.0, further confirming that R37J has extremely low similarity to known phage genomes, belonging to a completely new phage. Figure 3 c).

[0058] The bacteriophage R37J was named V. alginolyticus phage vB_ValM-R37J and was deposited on August 6, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, 510070, China, with accession number 66813-B1.

[0059] 3.5 Host range of bacteriophage R37J

[0060] This study tested the host range of 101 bacterial strains, including 89 Vibrio strains from diverse sources. These strains encompassed 14 Vibrio species isolated from aquaculture, marine, and public health-related environments. The results showed that bacteriophage R37J could infect 46 of these Vibrio strains, achieving an infection rate of 51.7%. Figure 4 These infectible strains were distributed across nine Vibrio species, including *V. alginolyticus* (28 strains), *V. parahaemolyticus* (8 strains), *V. owensii* (1 strain), *V. campbellii* (2 strains), *V. harveyi* (2 strains), *V. splendidus* (1 strain), *V. tubiashii* (2 strains), *V. brasiliensis* (2 strains), and *V. metschnikovii* (1 strain). The isolation sources included diseased animals, healthy animals, water bodies, biofilms, and clinical samples, indicating that R37J possesses strong cross-species infectivity. Specifically, R37J achieved a 65.4% infection rate against Vibrio bacteria in aquaculture environments, infecting a total of 34 strains, demonstrating its broad-spectrum and highly efficient lysis potential in aquaculture.

[0061] Building upon this foundation, this study further evaluated the lytic ability of R37J against host bacteria using a dilution titration method. The study found that R37J can form both "spots" and "plaques" on host bacterial colonies, representing different lytic strategies. The lytic strategy that simultaneously forms spots and plaques is the endogenous lytic strategy (Lysis from within, LI), while the lytic strategy that only forms spots is the exogenous lytic strategy (Lysis from without, LO) (Abedon, 2011). R37J exhibits significantly better bactericidal effects on endogenous lytic hosts than on exogenous lytic hosts, and it can only proliferate through endogenous lytic hosts. Endogenous lytic hosts are distributed across multiple Vibrio species, without showing species concentration, suggesting that R37J has the ability to lyse across species. Notably, seven of these strains originated from the culture environment, demonstrating R37J's strong bactericidal ability against culture-related Vibrio bacteria. Overall, bacteriophage R37J not only has a broad host range but also exhibits diverse and regular bactericidal strategies on different host bacteria.

[0062] 3.6 Environmental stability of bacteriophage R37J

[0063] The results of the thermal stability test showed that bacteriophage R37J could maintain a high survival rate (>80%) within a certain time (3 h) in the range of 4℃ to 37℃, indicating that it has good adaptability to the common environmental temperature of aquaculture. Figure 5 a). When the temperature rises above 37℃, especially at 45℃ and higher, the activity of bacteriophages decreases rapidly, and prolonged treatment time leads to further inactivation. pH stability experiments show that bacteriophage R37J maintains a high survival rate (>70%) within the pH range of 6–9, indicating good stability within the common fluctuation range of aquaculture water (pH 6.5–8.5). Figure 5 (b) However, under extreme acidic or alkaline conditions (pH ≤ 4 and pH ≥ 11), phage R37J becomes inactive within a short period. In summary, phage R37J exhibits good physicochemical stability within the temperature and pH range commonly found in aquaculture, maintaining continuous activity and infectivity after release. However, appropriate low-temperature preservation measures are necessary during high summer temperatures or during transportation and storage to prevent excessively high temperatures from causing phage inactivation and affecting its application effectiveness.

[0064] 3.7 Bactericidal effect of bacteriophage R37J

[0065] The lytic activity of bacteriophage R37J against the host bacterium V. alginolyticus D61_T3_15_W4 under different MOI conditions is shown in the figure. Figure 6 In the early stages of infection (0-3 h), R37J rapidly inhibited host bacterial growth, exhibiting significant and strong lytic activity (>62%) under all MOI conditions. The bactericidal efficiency increased with increasing MOI, with the high MOI group showing even faster and more efficient inhibition of host growth. This ability to rapidly control host growth indicates that R37J possesses a significant "preemptive inhibition" advantage in the early stages of infection. Around 8 h after phage addition, the phage lytic efficiency reached its peak in each MOI group, with an average bactericidal efficiency as high as 85%, demonstrating a strong bactericidal effect, and maintaining a good and sustained antibacterial effect for a period afterward. Although the OD in the later stages of culture... 600 A slight rebound was observed, but its bactericidal efficiency remained above 40%. These results indicate that bacteriophage R37J can not only rapidly and efficiently lyse the host bacterial population for effective early control, but also maintain its antibacterial ability for a relatively long period, demonstrating good sustained control characteristics.

[0066] 3. Discussion

[0067] Successful phage therapy relies on the efficient recognition, infection, lysis, and amplification of phages within pathogenic bacteria. Therefore, assessing their adsorption capacity, latency, and lysis rate is crucial. Rapid adsorption is key to efficient phage infection. Studies have shown that most phages achieve 70-90% adsorption within 5 minutes. For example, phage PCEc3 achieved 87% adsorption at 5 minutes and 95% at 20 minutes (Costa et al., 2025). Phage R37J achieved 93.7% adsorption after 5 minutes of contact with the host bacterium, further increasing to 99.5% after 10 minutes, demonstrating its strong initial recognition and attachment capabilities. In the natural environment, rapid adsorption not only helps phages preferentially occupy receptor sites on the host surface, blocking the invasion of other competing phages, but also allows them to complete adsorption before the host initiates defense mechanisms (such as secreting extracellular polymers or modifying receptors), thereby improving overall infection efficiency (Wang et al., 2023). Most reported myotail phages have a latency period of 20-30 min and a lysis rate of 50-200 cells. -1 In lytic phages, there is typically a "quantity-rate" trade-off between lysis rate and latency. A short latency promotes rapid amplification but limits viral particle replication time, while a longer latency provides more time for replication and assembly, leading to higher lysis rates (Abedon et al., 2003). For example, the myotail phage vB_VpaM_R16F, which infects Vibrio parahaemolyticus, has a latency of 10 min and a burst rate of only 13 PFU / cell, a typical example of a phage following a short latency and low burst rate survival strategy (Chen et al., 2023). In contrast, the myotail phage XZB, which infects Vibrio alginolyticus, has a latency of approximately 40 min and a burst rate as high as 570 PFU / cell. -1(Gao et al., 2024). Phage R37J exhibits an infection strategy characterized by rapid adsorption, moderate latency, and high burst volume. It can efficiently attach to host cells in a short time, ensuring the rapid initiation of subsequent lysis cycles. Although its latency is slightly longer, this allows for more complete genome replication and assembly within the host cell, achieving a lysis rate of up to several hundred viral particles, thereby enhancing its transmission efficiency within the host population and creating a typical "therapeutic amplification effect." This survival strategy is particularly advantageous for therapeutic applications: it can efficiently identify and kill pathogenic bacteria in a short time, followed by a rapid increase in local viral concentration through a high burst volume. The genomic mechanism of phage R37J further supports its high lysis performance. Its encoded highly efficient lysis-related enzymes, such as Holin and Peptidase M15, work synergistically to precisely control the timing and intensity of host membrane and cell wall degradation, significantly improving lysis efficiency and viral particle release. Therefore, phage R37J, with its rapid adsorption capacity, high lysis rate, and synergistic support from multi-level molecular mechanisms, exhibits strong infection and amplification potential, making it a promising candidate for therapeutic phages.

[0068] Host range experiments showed that R37J could effectively lyse 41 strains of 8 out of 14 Vibrio species, achieving a coverage rate of 51.7%, with an infection rate of 65.4% among aquaculture-related strains, demonstrating its strong adaptability to dominant pathogens in the aquaculture environment. This broad-spectrum infectivity provides significant advantages for its practical application in aquaculture systems: on the one hand, phage R37J can simultaneously target multiple common Vibrio pathogens (such as V. alginolyticus, V. parahaemolyticus, etc.) as well as some potential emerging pathogens (such as V. brasiliensis, V. tubiashii, etc.), enhancing its practicality and reliability in dealing with mixed infections of multiple pathogens or dynamic pathogen turnover in aquaculture systems; on the other hand, the broad host range also significantly reduces dependence on mixtures of multiple phages, helping to simplify the formulation and application of therapeutic agents, thereby improving practical operability and reducing costs. Furthermore, phage R37J exhibited effective lysis against strains from multiple sources (disease-stricken animals, water bodies, clinical samples, etc.), suggesting that it may be able to recognize certain highly conserved or functionally critical receptor structures, thereby maintaining stable and widespread infectivity in complex aquaculture environments. Combined with structural prediction analyses using AlphaFold and Foldseek, six trimeric fibrillary proteins (ORF 62, 63, 65, 69, 71, and 158) were identified in its genome. This structural feature provides an important molecular basis for its cross-species host recognition ability. These diverse tail fibrillary proteins may synergistically participate in the recognition process of different host surface receptors, constructing the broad host-range infection characteristics of phage R37J. Combined with its high lysis efficiency and burst capacity, R37J holds promise as a therapeutic phage for single-strain use, achieving rapid onset, broad-spectrum coverage, and easy-to-use precise sterilization effects in aquaculture.

[0069] Meanwhile, R37J also offers advantages in terms of genetic safety. Genomic analysis did not detect virulence genes, antibiotic resistance genes, or integrase elements related to lysogenicity, indicating that it is a strictly lytic phage, avoiding the biosafety risks associated with horizontal gene transfer. Its genome also carries various AMGs, such as functional genes related to nucleotide synthesis, carbon metabolism, and cell protection, which may endow the phage with the ability to regulate host metabolism and optimize its own replication environment during infection, thereby improving its adaptability and replication efficiency under different physiological states.

[0070] Bacteriophage R37J exhibits excellent thermal stability over a wide temperature range of 4°C to 37°C and maintains high activity within a pH range of 6–9, covering most aquaculture water conditions. This allows it to adapt to different seasons and the daily operating conditions of aquaculture systems. This environmental adaptability ensures that it can continuously complete the rapid adsorption and lysis infection cycle during actual use, providing a stable and reliable viral source for controlling pathogens. Especially in aquaculture, R37J can maintain its activity for a long time under the normal physicochemical conditions of aquaculture water, meaning that it can maintain its control effect without frequent replenishment. This promises to achieve a low-frequency, high-efficiency control application model, reducing management costs. The sensitivity of bacteriophage R37J to extreme temperatures and pH values ​​also suggests that a reasonable low-temperature cold chain system should be configured in the storage and transportation process to ensure its activity stability before and after actual use. This provides a clear process reference and risk avoidance direction for future formulation development and field promotion. The high adaptability and clear operational boundaries of bacteriophage R37J in conventional aquaculture environments lay a solid foundation for its use as a bacteriophage formulation specifically for aquaculture and are important supporting characteristics for promoting its commercialization.

[0071] The advantages of bacteriophage R37J in terms of lysis strategy, host range, genomic safety, and environmental stability comprehensively lay the foundation for its broad application prospects in phage therapy for aquaculture. Bactericidal efficiency experiments further validated its practical application effects. The results showed that R37J can not only achieve rapid and efficient lysis of the host bacterial population in the early stages, but also maintain significant antibacterial activity for a relatively long period, demonstrating excellent sustained control capabilities. This sustained antibacterial effect is a direct manifestation of its highly efficient infection strategy, indicating that R37J not only possesses strong initial lysis capabilities but can also establish a stable control advantage in the aquaculture system, thereby effectively curbing the rebound and regeneration of pathogens, providing solid support for precise and efficient phage therapy.

Claims

1. Bacteriophage 溶藻弧菌 Phage vB_ValM-R37J, with accession number GDMCCNO.66813-B1.

2. A microbial preparation for controlling various Vibrio infections in aquaculture environments, characterized in that, Contains the bacteriophage according to claim 1 溶藻弧菌 Phage vB_ValM-R37J is used as the active ingredient.

3. The bacteriophage according to claim 1 溶藻弧菌 The use of the microbial preparation of phage vB_ValM-R37J or claim 2 in the prevention and / or eradication of Vibrio, for non-disease diagnostic and therapeutic purposes, wherein the Vibrio is 溶藻弧菌 , 副溶血性弧菌 , 奥氏弧菌 , 坎氏弧菌 , V. 哈维氏弧菌 , 灿烂弧菌 , 塔氏弧菌 , 梅氏弧菌 and / or 巴西弧菌 .

4. The bacteriophage according to claim 1 溶藻弧菌 The application of the microbial preparation described in phage vB_ValM-R37J or claim 2 in the preparation of products for controlling Vibrio infection in aquaculture, wherein the Vibrio is V. 溶藻弧菌 , 副溶血性弧菌 , 奥氏弧菌 , 坎氏弧菌 , 哈维氏弧菌 , V. 灿烂弧菌 , 塔氏弧菌 , 梅氏弧菌 and / or 巴西弧菌 .

5. The application according to claim 4, characterized in that, The products described above include pharmaceuticals, disinfectants or water purifying agents.

6. A product for controlling Vibrio infection in aquaculture, characterized in that, Contains the bacteriophage according to claim 1 V. 溶藻弧菌 Phage vB_ValM-R37J or the microbial preparation according to claim 2.

7. A method for the in vitro prevention and treatment of harmful Vibrio infections for non-disease diagnostic and therapeutic purposes, characterized in that, It utilizes the bacteriophage described in claim 1 溶藻弧菌 The microbial preparation described in Phage vB_ValM-R37J or claim 2 kills Vibrio in vitro through its lytic effect on Vibrio, wherein the Vibrio is... 溶藻弧菌 , V. 副溶血性弧菌 , 奥氏弧菌 , 坎氏弧菌 , 哈维氏弧菌 , 灿烂弧菌 , V. 塔氏弧菌 , 梅氏弧菌 and / or 巴西弧菌 .

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