A lytic bacteriophage for preventing and controlling vibrio in aquaculture environment

Abstract

This invention discloses a lytic bacteriophage for controlling Vibrio alginolyticus in aquaculture environments. The bacteriophage, vB_ValM_R42H, belongs to the myotail bacteriophage family, is insensitive to chloroform, has a lipid-free capsid, and its preservation number is GDMCC No:67434-B1. This bacteriophage exhibits rapid adsorption, short latency, and high lysis rate, and maintains high activity over a wide temperature range (4℃~55℃) and pH range (2~11), demonstrating strong environmental adaptability. Genomic analysis shows that this bacteriophage does not contain virulence factors, antibiotic resistance genes, or lysogen-related genes, indicating high biosafety. Phylogenetic analysis indicates that it can be classified into a new genus. Due to its high lysis efficiency, good environmental stability, strong specificity, and genetic safety, the bacteriophage R42H of this invention can serve as an ideal biocontrol agent for controlling Vibrio alginolyticus disease in aquaculture.

Description

A lytic bacteriophage for controlling Vibrio in aquaculture environments Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a lytic bacteriophage for controlling Vibrio in aquaculture environments. Background Technology

[0002] With the continuous expansion of the aquaculture industry, changing farming methods is an inevitable choice to increase output and value. Intensive farming is a technology that can significantly improve farming efficiency, featuring high yield, low cost, and ease of management. However, its increasing scale year by year has also led to a significant increase in the frequency of disease outbreaks in farmed organisms, resulting in huge economic losses. Although people can reduce the risk of disease through various means, such as maintaining water quality, controlling the temperature and salinity of the aquaculture water, reducing stocking density, and improving the sanitary conditions of the aquaculture environment, these preventive measures have little effect and fail to meet expectations. Pathogens that cause diseases in aquatic animals mainly include bacteria, viruses, algae, parasites, and fungi. Among them, bacteria cause the greatest harm to aquatic animals. According to statistics, about 34% of diseases in aquatic animals are caused by bacteria. Vibrio disease in aquatic organisms is the most serious threat among bacterial infectious diseases, severely affecting the normal growth and development of farmed organisms. It also brings huge economic losses to farmers and has a detrimental impact on water environment safety, aquatic product quality, and human health.

[0003] Currently, the prevention and control of bacterial diseases mainly rely on chemical drugs such as antibiotics. However, the extensive use of antibiotics has inevitably caused many harms. Not only do they lead to drug resistance in target organisms, but they also leave excessive residues in the aquatic environment (such as tetracyclines and fluoroquinolones), causing bioaccumulation and toxicity in non-target organisms. Antibiotic residues and the spread of antibiotic resistance genes (ARGs) are prevalent in aquaculture and can also be transmitted to humans through the food chain, posing a significant threat to human health. Traditional antibiotic control is currently trapped in a vicious cycle of "ineffective treatment - increased dosage - increased resistance." Antibiotic resistance is gradually becoming an increasingly serious global health problem, and the World Health Organization, in its first global report on antibiotic resistance, emphasized the dangers of the impending post-antibiotic era. On the other hand, because antibiotics are less profitable than those in other markets, pharmaceutical companies are showing declining interest in developing and producing new antibiotics, making the search for green and efficient alternatives for prevention and control an urgent priority.

[0004] Bacteriophages are a class of viruses that specifically lyse bacteria. They are ubiquitous in any environment containing their bacterial hosts and play a vital role in many biological processes, making them considered the most abundant organisms on Earth. Based on their life cycle, bacteriophages are generally classified into virulent and temperate phages. Virulent phages initiate a lytic cycle, attaching themselves to their bacterial host, injecting their genome, replicating by overriding the host's molecular mechanisms, and finally lysing the host cell while releasing their progeny. Temperate phages, on the other hand, infect the host by initiating a lysogenic cycle. In this cycle, the phage genome remains dormant, replicating alongside the host, and occasionally entering a lysis cycle under specific triggers. Lysogenic phages are a crucial foundation for treating bacterial infections and are among the most promising alternative approaches currently available. In recent years, research on phage therapy in the field of pathogenic bacteria in aquaculture has been on the rise. Both the publication of research findings (papers, patents, etc.) and the sequencing of pathogenic phages in aquaculture environments have shown a significant upward trend, reflecting the broad interest and in-depth exploration of phage therapy for the prevention and control of bacterial diseases in aquaculture. For example, Viond et al. isolated and purified a lytic phage of Vibrio harveyi from a shrimp farming environment on the Indian coast. In phage therapy experiments, the survival rate of the experimental group was significantly higher than that of the control group without phage, and its effect was far superior to that of the antibiotic-treated group. This demonstrates that phages, as natural bactericidal substances, are more effective than antibiotics (Vinod et al., 2006). Considering the resistance of pathogens to bacteriophages, Mateus et al. used Vibrio parahaemolyticus bacteriophages VP-1, VP-2, and VP-3 to explore the effectiveness of the bacteriophage cocktail method. The results showed that the combined use of three bacteriophages was most effective in controlling Vibrio parahaemolyticus, maintaining the abundance of Vibrio parahaemolyticus at the lowest level in the experimental group (Mateus et al., 2014). A recent study indicated that bacteriophages can reside on mucosal surfaces, providing external protection against bacterial infection in yellow catfish, which enhances the interaction between fish, bacteria, and bacteriophages (Wu et al., 2024). Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a lytic bacteriophage vB_ValM_R42H and its application in the prevention and control of Vibrio diseases in aquaculture environments.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first objective of this invention is to provide a lytic bacteriophage vB_ValM_R42H, with accession number GDMCC No: 67434-B1.

[0008] This invention isolates and identifies a novel lytic bacteriophage R42H from a shrimp farming pond in Xiangshan County, Ningbo City, Zhejiang Province. The morphology and taxonomic position of this bacteriophage were determined through morphological observation, whole-genome sequencing, phylogenetic analysis, and comparative genomic analysis. It was named Vibrio alginolyticusphagevB_ValM_R42H and deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC No: 67434-B1, located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China.

[0009] A second object of the present invention is to provide the use of the above-mentioned bacteriophage vB_ValM_R42H in the preparation of products for inhibiting or killing Vibrio alginolyticus.

[0010] Preferably, the product is a bactericide, bacteriostatic agent, or biocontrol agent.

[0011] Preferably, the product is a disinfectant or water purifier for use in aquaculture environments.

[0012] Preferably, the product is a pharmaceutical composition for the prevention or treatment of Vibrio alginolyticus infections in aquatic animals.

[0013] Preferably, the product is an animal feed additive, feed premix, or drinking water agent.

[0014] A third object of the present invention is to provide a composition for inhibiting or killing Vibrio alginolyticus, comprising an effective amount of bacteriophage vB_ValM_R42H and a pharmaceutically or aquaculture-acceptable carrier, excipient or diluent.

[0015] Preferably, the dosage form of the composition is a liquid, wettable powder, granules, lyophilized powder for injection, or microcapsule.

[0016] A fourth objective of this invention is to provide a method for inhibiting or killing Vibrio alginolyticus, comprising applying an effective amount of bacteriophage to Vibrio alginolyticus or its possible environment.

[0017] Preferably, the application step is carried out by water splashing, soaking in a medicated bath, spraying, oral administration with feed, or injection.

[0018] In the process of intensive aquaculture, the economic losses caused by vibriosis and the drug resistance crisis resulting from antibiotic overuse have become core bottlenecks in the industry's development. Bacteriophages, as "natural enemies of bacteria," possess characteristics such as lytic activity, lack of drug resistance risk, and environmental stability, making them key candidates for green control. Biological and genomic characteristics are crucial for assessing the suitability of bacteriophages and determining their applicability for phage therapy. Several general properties should be considered, including bactericidal ability, bactericidal efficiency, and whether the bacteriophage carries endotoxins. To fit the in-situ survival environment, this study isolated a lytic bacteriophage, R42H, from a shrimp farming pond in Xiangshan, Ningbo. The core premise for bacteriophages as biocontrol agents is "no safety risk." VFDB and CARD analyses did not predict any antibiotic resistance genes or virulence factor-related genes in the genome of the R42H bacteriophage, indicating that infection with R42H will not lead to increased vibriosis or contaminate the aquaculture environment with antibiotic resistance genes.

[0019] The infection efficiency of bacteriophages is essentially a comprehensive reflection of their "molecular interactions" with the host bacteria. R42H exhibits characteristics of "nearly 100% adsorption rate at 5 min, a latency period of 10 min, and a lysis rate of 91 PFU / Cell." In contrast, previously reported bacteriophages typically have a latency period of 15–30 min and an outbreak size of 20–100 PFU / Cell (Chen et al., 2023; Ding et al., 2020; Ye et al., 2022), indicating that R42H has superior lysis capabilities. A trade-off exists between latency and lysis rate during the proliferation of virulent bacteriophages. The core mechanism lies in the fact that when bacteriophages release progeny, they disrupt the molecular mechanisms within the host cell used to produce more progeny. Therefore, bacteriophages that sacrifice some lysis in exchange for a short incubation period can be regarded as highly efficient proliferating specialized strains in environments with high bacterial incidence. This characteristic enables them to respond quickly to host population outbreaks and rapidly suppress bacterial numbers through a cycle of "lysis-proliferation-reinfection," which is highly consistent with the characteristics of bacteriophage R42H in this study, further confirming the application advantages of this bacteriophage in emergency prevention and control of vibrio disease in aquaculture.

[0020] Within the structural modules of the R42H bacteriophage genome, numerous tail-related proteins were predicted, including ORF24 (tail sheath), ORF26 (tail fiber protein), ORF28 (tail assembly chaperone), and ORF36 (baseplate wedge subunit). It has been reported that the host range of bacteriophages is related to tail fibers or receptor-binding proteins. The specific structures that bacteriophages use to target bacterial cells when infecting their hosts are determined by the specific structures they employ. Tail phages utilize various receptor-binding proteins, such as tail fibers, tail spikes, and central tail processes, to target their associated bacterial cell surface receptors. The ability of bacteriophage R42H to infect 10 strains of Vibrio alginolyticus with similar serotypes can also be attributed to the structural specificity of tail fibers, consistent with the common pattern that "sequence differences in tail-related proteins dominate host range differentiation."

[0021] In recent studies, phage-encoded endolysins have been shown to be effective in controlling foodborne pathogens, including Gram-negative bacteria, and in clearing bacterial biofilms. Notably, R42H identifies two endolysin genes. Produced in the late stages of phage replication, these genes break down bacterial peptidoglycan in the host cell wall from within, leading to the lysis of the host bacteria and the release of newly assembled phage particles. ORF66 (Endolysin) and ORF67 (N-acetylmuramoyl-L-alanine amidase) form a "dual lysis system," where the endolysin is responsible for degrading the cell wall peptidoglycan backbone, while the amidase targets the MurNAc-L-alanine amide bond. This synergistic effect enhances its lytic ability against biofilm-forming Vibrio. The translucent halo surrounding the plaque of R42H also confirms the presence of a polysaccharide depolymerase, and predictions using Phage AI software further confirm that phage R42H possesses a 100% lysis potential.

[0022] Furthermore, the lysis curves reveal the significant application potential of bacteriophages in treating Vibrio diseases in aquaculture. Bacteriophage R42H exhibits strong lysis against host Vibrio bacteria, making it suitable for phage therapy. It can effectively control the abundance of host Vibrio bacteria in the early stages, thus controlling Vibrio diseases. We evaluated the bacterial inactivation effect under different MOI conditions. The results showed that bacteriophage R42H has a highly efficient inactivation effect in preventing and controlling Vibrio alginolyticus. The "rapid response-sustained inhibition" characteristic of R42H is particularly prominent. At 8 h, the lysis efficiency in the MOI=0.001 group reached 83%, and after 12 h, it still maintained an inhibition rate of over 40%, indicating that even at low doses, R42H can continuously suppress Vibrio populations through the amplification effect of "lysis-proliferation-re-lysis." This characteristic can effectively address the "explosive growth" of Vibrio in the aquaculture environment, avoiding the dilemma of antibiotics being "effective during use but rebounding after discontinuation."

[0023] The results of thermal and pH stability experiments show that R42H exhibits high activity within the range of 4–55℃ and pH 2–11. When applying bacteriophages to aquaculture environments, the preparation process, storage conditions, transportation process, and actual environmental conditions must be considered. Excessively high temperatures or pH fluctuations may affect phage activity and therapeutic efficacy. Some studies have demonstrated that the thermal and pH stability of phages varies depending on the phage strain. Considering the typical conditions for shrimp farming in my country (water temperature 15–32℃, pH 7.5–8.5), the stability range of R42H is fully covered, and its environmental stability results provide a reference for the production, preparation, sales, transportation, storage, and optimal use of phages.

[0024] Compared with the prior art, the present invention has the following significant advantages:

[0025] 1. High lysis efficiency: Bacteriophage R42H has a fast adsorption rate (nearly 100% adsorption rate in 5 minutes), a short latency period (10 minutes), and a large lysis rate (91 PFU / cell), which can quickly and efficiently kill host bacteria.

[0026] 2. Strong environmental adaptability: It maintains high activity in a temperature range of 4℃ to 55℃ and a pH range of 2 to 11, and can adapt to complex and ever-changing aquaculture environments, making it widely applicable.

[0027] 3. High biosafety: The genome does not contain virulence factors, antibiotic resistance genes, or lysogen-related genes, ensuring its safety as a biocontrol agent and avoiding potential ecological risks.

[0028] 4. Broad application prospects: This bacteriophage exhibits high specificity against Vibrio alginolyticus, making it an ideal biocontrol agent for Vibrio alginolytic disease in aquaculture. In the future, it can be used in combination with other bacteriophages to develop a "phage cocktail," thereby expanding the scope of pathogen control and effectively delaying the development of bacterial resistance.

[0029] The Vibrio alginolyticusphage vB_ValM_R42H of the present invention was deposited on December 8, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No: 67434-B1, at the address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. Attached Figure Description

[0030] Figure 1 shows the morphological characteristics and chloroform sensitivity of bacteriophage R42H. (A) Plaques of bacteriophage R42H; (B) Electron micrograph of bacteriophage R42H; (C) Chloroform sensitivity of bacteriophage R42H.

[0031] Figure 2 shows the adsorption efficiency of bacteriophage R42H and its one-step growth curve. (A) Adsorption efficiency of bacteriophage R42H; (B) One-step growth curve of bacteriophage R42H.

[0032] Figure 3 shows the host range and phylogenetic tree of bacteriophage R42H. "Red dots" indicate that the bacteriophage can infect this bacterium.

[0033] Figure 4 shows the bactericidal effect of bacteriophage R42H. (A) Lysis curve of bacteriophage R42H; (B) Lysis efficiency of bacteriophage R42H.

[0034] Figure 5 shows the environmental stability of bacteriophages. (A) Survival rate of bacteriophage R42H at temperatures ranging from 4℃ to 65℃; (B) Survival rate of bacteriophage R42H at pH values ​​ranging from 2 to 13.

[0035] Figure 6 shows the complete genome map of bacteriophage R42H.

[0036] Figure 7 shows the phylogenetic analysis and whole-genome alignment of bacteriophages. (A) Phylogenetic tree of bacteriophage R42H; (B) Viridic heatmap of bacteriophage R42H; (C) Whole-genome alignment of bacteriophage R42H. Detailed Implementation

[0037] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0038] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are commercially available. All quantitative experiments in the following embodiments were performed in triplicate, and the results were averaged.

[0039] Example 1: Isolation, purification and enrichment of bacteriophages

[0040] 1. Method

[0041] 1.1 Isolation and purification of bacteriophages

[0042] This study used *V. alginolyticus* NB2303_I4W as the host for phage isolation. This strain was a wild-type strain, isolated from the hepatopancreas and intestinal tissue of shrimp exhibiting typical vibriosis symptoms. The phage was preserved long-term using the glycerol cryopreservation method. Fresh bacterial culture was mixed with sterile 50% glycerol at a 1:1 volume ratio, aliquoted into sterile cryovials, and stored at -80°C. Water samples were collected from the top 50 cm of a shrimp farming pond in Xiaoyangjia Village, Xiangshan City, Ningbo, Zhejiang Province (121.83°E, 29.39°N) in the summer of 2023 for phage isolation. The water samples were filtered through a 0.22 μm polycarbonate membrane (Polycarbonate, Millipore, USA) and stored in the laboratory at 4°C.

[0043] Before the isolation experiment, 1 mL of the host bacterium (V. alginolyticus NB2303_I4W) preservation solution was added to 50 mL of 2216E liquid medium (10 M tryptone, 2 M yeast extract, artificial seawater at pH 7.5, autoclaved at 110℃ for later use). The medium was then incubated at 28℃ and 160 rpm in a shaker for 3-4 h to allow the bacterial culture to reach the early logarithmic growth stage (OD). 600=0.1-0.2), and then the filtered water sample was added to the V. alginolyticus NB2303_I4W bacterial culture in the early logarithmic growth stage. After overnight incubation on a shaker (160 rpm, 28℃), the culture was filtered to remove bacteria and enrich potential phages. The co-culture solution was centrifuged at 8,000×g, 4℃ for 10 min, and filtered through a 0.22 μm filter membrane to collect the supernatant, removing residual cells and impurities to obtain phage concentrate. 500 μL of logarithmic growth stage host bacterial culture was mixed with 7 mL of melted upper medium (2216E liquid medium + 0.5% agar powder) and poured into a lower medium (2216E liquid medium + 1.5% agar powder) plate and allowed to solidify. 5 μL of phage concentrate was added to the plate and incubated overnight at 37℃. The appearance of clear phage plaques indicates the presence of phages in the sample. Then, the phages were isolated using the double-layer plate method. 100 μL of the concentrated phage solution was taken and serially diluted 10-fold (to a final concentration) with 900 μL of SM buffer (prepared by adding 5.844 g NaCl, 2.4647 g MgSO4·7H2O, 6.057 g Tris, and 0.100 g Gelatin to 1 L of ultrapure water; after preparation, the pH was adjusted to 7.50, and the buffer was autoclaved at 110℃ for 30 min and cooled before use). -6 Then, mix each of the phages separately with 1 mL of logarithmic-phase host bacterial culture, adsorb at room temperature in the dark for 10 min, add to 7 mL of 2216E medium containing 0.5% agar, and then pour the mixture into 2216E medium containing 1.5% agar. After the upper layer solidifies, seal the edges of the plate with sealing film, invert it and incubate at 28°C, observing periodically for plaque forming units (PFUs). Pick individual plaques for at least 3 rounds of purification until the morphology and size of the plaques formed on the plate are consistent. Then, pick the plaques and store them in 1 mL of SM buffer for subsequent use to obtain the purified phage stock solution, which is stored at 4°C in the dark.

[0044] 1.2 Enrichment of bacteriophages

[0045] The purified phage was inoculated into 50 mL of host bacterial culture in the early logarithmic growth stage. After the culture medium became clear or showed signs of lysis such as flocculent formation, cell debris was removed by centrifugation (8,000 × g, 4 °C, 15 min). The supernatant was filtered through a 0.22 μm filter and then inoculated into a 200 mL system. The above steps were repeated to gradually expand the system to 1 L. Then, DNase I and RNase A were added to a final concentration of 1 μg / mL for digestion at room temperature for 1 h. NaCl was then added to a final concentration of 1 mol / L, and after complete dissolution, the mixture was placed at 4 °C for about 1 h to promote the detachment of phage fragments into the suspension. The supernatant was collected by centrifugation (10,000 × g, 4 °C, 15 min) and filtered through a 0.22 μm filter to obtain 1 L of phage filtrate. Add PEG 8000 to the phage fluid to a final concentration of 10% (wt / vol), and incubate at 4°C in the dark for 1–3 days to allow phage particles to aggregate and precipitate. Collect the precipitate by centrifugation at 12,000 × g for 1 h and resuspend it in approximately 6 mL of SM buffer. Prepare CsCl solutions with final concentrations of 1.3 g / mL, 1.5 g / mL, and 1.7 g / mL in advance using SM buffer to suspend the resuspended phages (abundance not less than 10⁻⁶). 10 The phage concentrate (PFU / mL) was ultracentrifuged, with 2 mL added sequentially to each centrifuge tube. Finally, the concentrated phage solution was added to the top layer of the CsCl solution, and the mixture was ultracentrifuged at 200,000 ×g for 24 h at 4 °C. The blue concentrated phage band was then removed, and CsCl was removed by ultrafiltration at 5,000 ×g at 4 °C using a 30 kDa ultrafiltration tube. The phage was washed with SM buffer, transferred to centrifuge tubes, and stored at 4 °C.

[0046] 2. Results

[0047] In this study, a bacteriophage, vB_ValM_R42H (hereinafter referred to as R42H), was isolated from the surface 50 cm water (121.83°E, 29.39°N) of a shrimp farming pond in Xiaoyangjia Village, Xiangshan County, Ningbo City, Zhejiang Province, using V. alginolyticus NB2303_I4W as the host. Using the double-layer plate method, rapid plaque formation was observed (Figure 1, A). Clear, translucent circular plaques approximately 1 mm in diameter were visible to the naked eye after about 6 hours of incubation. The plaque diameter gradually increased in size and translucency with prolonged incubation. Almost all Vibrio bacteria in the plaque area were lysed, reflecting the extremely strong lytic activity of bacteriophage R42H. Furthermore, each plaque was surrounded by a semi-transparent halo (Figure 1, A), which is often considered to be related to the polysaccaride depolymerase within the bacteriophage.

[0048] Example 2: Morphological observation of bacteriophages

[0049] 1. Method

[0050] Phage morphology was observed using TEM with phosphotungstic acid negative staining. 5 µL of phage suspension was placed on a 200-mesh copper grid and allowed to adsorb in the dark for 30 min. The phage sample was negatively stained with 1% phosphotungstic acid solution (pH=7.0) for 20 min, and then dried for 30 min. The sample was observed using a JEM-2100 transmission electron microscope at an accelerating voltage of 80 kV. Finally, ImageJ image analysis software was used to process the acquired images and measure the size and morphological characteristics of the phages.

[0051] 2. Results

[0052] TEM morphology results showed that the head of bacteriophage R42H was an icosahedron with a diameter of 67.62±1.04 nm, and its retractable tail was 93.34±1.41 nm, exhibiting a typical myotail bacteriophage morphology (B in Figure 1).

[0053] Example 3: Chloroform susceptibility assay of bacteriophages

[0054] 1. Method

[0055] The chloroform susceptibility test for bacteriophages is used to determine whether the bacteriophage contains lipids. This experiment included three parallel controls. One mL of bacteriophage with an infectivity of 3.8 × 10⁻⁶ was used. 11 Add 200 μL, 20 μL, and 0 μL of chloroform to the PFU / mL phage solution, respectively, mix well, and incubate at room temperature in the dark for 30 min. Centrifuge at 5,000 × g, 4°C for 5 min and collect the supernatant. Add 1 mL of logarithmic growth phase host bacterial culture and 7 mL of approximately 50°C 0.5% agar 2216E medium and mix quickly. Pour evenly onto 1.5% agar 2216E solid culture plates. After the plates solidify, seal the edges with sealing film and place in an incubator at 28°C.

[0056] 2. Results

[0057] The chloroform sensitivity test results (C in Figure 1) showed that there was no significant difference in the phage plaques titrated by different chloroform concentration treatment groups. The morphology, size, growth rate, and turbidity of the phage plaques were all consistent, indicating that phage R42H is not sensitive to chloroform, does not contain lipids in its capsid, and is not encapsulated by a lipid membrane.

[0058] Example 4: Determination of phage adsorption efficiency and one-step growth curve

[0059] 1. Method

[0060] 1.1 Determination of Adsorption Efficiency

[0061] Phage and bacterial suspension were added at an MOI of 0.01 and cultured on a shaker. At 0, 5, 10, 15, and 20 min after mixing, 1 mL of the mixture was centrifuged at 12,000 × g for 1 min. Then, 100 μL of the supernatant (unadsorbed free phage) was added to 900 μL of SM buffer for a 10-fold serial dilution, with three replicates. The titer was determined using the double-layer agar plate method. Based on the titer, the number of phages adsorbed onto the host bacteria was calculated. The experiment was repeated three times. The calculation formula is: Phage adsorption rate = (1 - Titer of unadsorbed phage / Initial phage titer) × 100%.

[0062] 1.2 One-step growth curve determination

[0063] To evaluate the infectivity and replication efficiency of bacteriophages, a one-step growth curve experiment was conducted to determine the optimal conditions for phage reproduction. This experiment followed the method of (Pajunen et al., 2000) with some modifications. The host bacterium *Vibrio alginolyticus* NB2303_I4W was inoculated at a 1:100 ratio in 2216E liquid medium and cultured on a shaker (160 rpm, 30℃) until the early logarithmic growth phase (OD). 600 =0.1-0.2). 500 μL of phage was added to 50 mL of host bacterial culture in the early logarithmic growth stage to achieve a multiplicity of infection (MOI) of 0.01. After adsorption in the dark for 5 min, the phage-host mixture was centrifuged (8,000 × g, 4℃, 4 min), the supernatant was discarded, and the precipitate was resuspended in 1 mL of 2216E liquid medium. This centrifugation was repeated twice to thoroughly remove any free phage not adsorbed to the host cells. Finally, 1 mL of the resuspended phage was inoculated into 50 mL of 2216E liquid medium and cultured on a shaker (160 rpm, 30℃) for one hour. Phage infectivity was determined every 10 min using a double-layer plate method. The outbreak size was calculated by measuring the ratio of phage counts immediately before and after progeny phage release, and a one-step growth curve was plotted based on the numerical values. Each treatment group was replicated in three places.

[0064] 2. Results

[0065] The latency and lysis rate of bacteriophages can be investigated based on adsorption efficiency and one-step growth curves. This includes the time required for a bacteriophage to adsorb, invade, assemble, and release progeny phages, as well as the average number of progeny phages released from each infected host cell. The adsorption efficiency of bacteriophage R42H (Figure 2, A) shows that the adsorption percentage rapidly increases to nearly 100% within 0-5 minutes, and then stabilizes after 5 minutes. This indicates that bacteriophage R42H adsorbs to host bacteria extremely rapidly, initiating the lysis process within 5 minutes. The one-step growth curve results (Figure 2, B) show that for the first 10 minutes or so, the PFU level of bacteriophage R42H remains low, corresponding to the adsorption process of the host bacteria; this process is called the "adsorption phase." For the next 10 minutes after the adsorption phase, the PFU level shows no significant change, corresponding to the process of the bacteriophage completing invasion, genome replication, and progeny assembly within the host cell; this process is called the "latency phase." After the latency phase, the PFU level rises rapidly, representing the rupture of the host cell and the release of progeny bacteriophages, reaching the "lysis phase." On average, each infected host cell releases 91 progeny bacteriophages. In summary, bacteriophage R42H exhibits rapid adsorption, a short latency period, and a high lysis rate (91 PFU / cell), demonstrating its high efficiency in infecting the host and strong proliferative capacity.

[0066] Example 5: Host lysis profile of bacteriophages

[0067] 1. Method

[0068] To determine the host lysis profile of bacteriophage vB_ValM_R42H, a total of 64 Vibrio strains were selected for experiments, including 41 Vibrio alginolyticus strains, 7 Vibrio harveyi strains, 5 Vibrio parahaemolyticus strains, and 11 other Vibrio strains (Vibrio cannii, Vibrio erwinii, Vibrio brasiliensis, etc.). The host range of the bacteriophage was determined using plaque titration. 1 mL of logarithmic growth phase host bacterial culture was rapidly mixed with 7 mL of 0.5% agar 2216E medium at approximately 50°C, and poured evenly onto 1.5% agar 2216E solid culture plates and allowed to solidify. 5 μL of bacteriophage liquid (infectivity approximately 3.8 × 10⁻⁶) pre-diluted with SM buffer was then used. 6 -10 11 A drop of PFU / mL was placed onto a cooled and solidified Vibrio agar plate and incubated at 30°C to observe plaque formation. Each treatment was performed in triplicate, with Vibrio alginolyticus NB2303_I4W used as a positive control instead of the test strain, and SM buffer used as a negative control.

[0069] 2. Results

[0070] The results (Figure 3) showed that bacteriophage R42H exhibited a wide host lysis range, infecting 10 of the 64 Vibrio strains tested, including V. alginolyticus I7, V. alginolyticus H10, V. alginolyticus I8Y, V. alginolyticus I4T, V. alginolyticus H8Y, V. alginolyticus H7, V. alginolyticus I4MI, V. alginolyticus I8W, V. alginolyticus I4W, and V. alginolyticus H8W. All 10 infectable Vibrio strains were Vibrio alginolyticus and had similar serotypes.

[0071] Example 6: Determination of bacteriophage bactericidal efficiency

[0072] 1. Method

[0073] To investigate the lytic activity of bacteriophage vB_ValM_R42H against the host Vibrio alginolyticus NB2303_I4W, bacteriocidal experiments were conducted under different MOI conditions. Vibrio alginolyticus NB2303_I4W was inoculated at a 1:50 ratio into 2216E liquid medium and cultured on a shaker (160 rpm, 30℃) until the early logarithmic growth phase (OD). 600 After adding bacterial culture medium and phage at MOIs of 0.1-0.2, bacterial culture and phage at different abundances were added according to MOIs of 0.01, 0.1, 1, 10, and 100 to obtain Vibrio-phage co-incubation systems. The absorbance of samples in each well was monitored in real time at 600 nm using a multi-mode microplate reader (Synergy H1, Bio-Tek, USA). Co-incubation was performed for 24 h, with measurements taken every 30 min. The plate was shaken for 5 s before detection at 28℃. Each treatment had at least five replicates, with 2216E liquid medium and bacterial culture medium serving as negative and positive controls, respectively. Data were analyzed using SPSS 21.0 statistical software, and all values ​​are expressed as mean ± standard deviation (SD). Statistical differences between treatments were assessed using one-way ANOVA and Duncan's multiple range test, with a significance level set at p < 0.05. Figures were generated using Prism 10.4.1 software.

[0074] 2. Results

[0075] Figure 4 shows the bactericidal results of bacteriophage R42H against the host *V. alginolyticus* NB2303_I4W under different MOI conditions. As can be seen from the figure, adding bacteriophage R42H to the host *V. alginolyticus* culture in the exponential growth phase rapidly lyses the host. Within 0-4 h post-infection, the OD600 values ​​of all bacteriophage-treated groups showed no significant difference from the bacterial control, indicating the "latent period." The OD600 value of the MOI=100 treatment group remained consistently around 0.2, showing the strongest inhibition. The OD600 value of the MOI=0.001 group increased slightly in the later stages, but remained far lower than the bacterial control. After 4 h, the increase in OD600 value of the bacteriophage-treated groups was significantly inhibited, and the degree of inhibition varied among different MOI groups. The bacteriophage exhibited the strongest bactericidal efficiency against Vibrio host bacteria at 8 h of culture, with a lysis efficiency reaching 83% at MOI=0.001. The OD600 value of the bacterial control continued to rise within 12-14 h, while the OD600 values ​​of all phage-treated groups remained relatively stable, indicating that the phage's inhibitory effect on host bacterial growth is "persistent." The lysis efficiency of all MOI groups showed a "rise then stabilize" trend with culture time: efficiency was generally low at 2 h, rapidly increased after 4 h, reached its peak at 8 h, and decreased slightly in some groups after 12 h, but still remained above 40%. This demonstrates that phage R42H has a significant and sustained lysis effect on host bacteria; the higher the phage MOI, the stronger the initial bacterial inhibition; and with increasing time, lower MOIs result in slower inhibition but a more sustained effect.

[0076] Example 7: Thermal stability and pH tolerance of bacteriophages

[0077] 1. Method

[0078] By testing the thermostability and pH sensitivity of bacteriophages, their ability to resist environmental stress can be investigated. The concentrated bacteriophage solution was first diluted with SM buffer to obtain 10... 8PFU / mL phage solutions were prepared for use. Temperature gradients of 4℃, 15℃, 25℃, 37℃, 45℃, 55℃ and 65℃ were set using a thermostat. The phage solutions were placed in incubators at different temperatures in the dark for 6 h. After the samples were brought to room temperature, the infectivity of the phages was determined using the double-layer plate method. Following the method proposed by Phumkhachorn and Rattanachaikunsopon in 2010 (Phumkhachorn et al., 2010), phage solutions were placed in SM buffer (prepared by adding 5.844 g NaCl, 2.4647 g MgSO4·7H2O, 6.057 g Tris, and 0.100 g Gelatin to 1 L of ultrapure water; after preparation, the pH was adjusted to 7.50, autoclaved at 110℃ for 30 min, and used after cooling). At room temperature, the pH of the system was adjusted to different gradients from 2 to 13 using NaOH and HCl. After 6 h of incubation in the dark at room temperature, the infectivity of the phages was determined using the double-layer plate method, with three replicates for each treatment. One-way ANOVA was used to assess statistical significance, followed by post-hoc multiple comparisons using IBM SPSS (v25.0) based on alphabetical grouping.

[0079] 2. Results

[0080] The environmental stability results of bacteriophage R42H are shown in Figure 5. It maintained high activity (survival rate >80%) within a temperature range of 4℃-37℃. The survival rate was close to 100% at 37℃, while high temperatures (≥45℃) significantly reduced its survival rate. At 45℃, the survival rate dropped to 60%, at 55℃ to 40%, and at 65℃, it was completely inactivated with a survival rate close to 0%, indicating that this bacteriophage is not tolerant of high temperatures (Figure 5, A). Furthermore, the optimal pH range for bacteriophage R42H is 5-10. It maintained high activity (survival rate >80%) in a near-neutral environment, but completely lost activity when the pH was above 13 (Figure 5, B). This characteristic matches the physiological environment of the host bacteria and is consistent with the characteristics of typical aquaculture environments, indicating that this bacteriophage can maintain high activity in the natural living environment of the host bacteria and can adapt to daily aquaculture environments, but it has weak tolerance to extreme temperatures and pH levels.

[0081] Example 8: Genomic Characterization of Bacteriophages

[0082] 1. Method

[0083] In this study, phage DNA was extracted using the phenol-chloroform extraction method. After digestion with 100 mg / mL proteinase K, 0.5 mol / L ethylenediaminetetraacetic acid (EDTA), and 10% sodium dodecyl sulfate (SDS) at 55°C for 3 h, phage DNA was extracted using the phenol / chloroform / isoamyl alcohol method. The specific steps are as follows: Add an equal volume of phenol / chloroform / isoamyl alcohol (volume ratio 25:24:1) to the digested and purified sample, mix well, and centrifuge at 4℃ and 12,000 ×g for 5 min to remove impurities. Transfer the upper aqueous phase to a new centrifuge tube, and repeat this step twice. Add an equal volume of chloroform / isoamyl alcohol (volume ratio 24:1) to the supernatant for further purification, and centrifuge at 4℃ and 12,000 ×g for 10 min. Then, add isopropanol to the supernatant to precipitate the precipitate and incubate overnight at -20℃. Wash the precipitate twice with pre-cooled 70% ethanol and air dry. Finally, dissolve the phage DNA in 100 μL TE buffer (10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH=8.0) and store at -80℃ for sequencing.

[0084] The phage whole-genome sequencing was performed by Shanghai Hanyu Biotechnology Co., Ltd. (China). The sequencing library was prepared by NEBNext® Ultra. TM DNA library preparation kits (NEB, USA) were used for construction, and sequencing was performed using the Illumina HiSeq 4000 platform. Raw data and genome assembly were analyzed using Trimmomatic v0.32 and Velvet v1.2.03 software.

[0085] 2. Results

[0086] To assess the biosafety of bacteriophage R42H in aquaculture biocontrol, a comprehensive genomic identification was performed. Bacteriophage R42H possesses linear double-stranded DNA with a total length of 42,752 bp and a G+C content of 42.96%. GenemarkS was used to predict the ORFs of bacteriophage R42H; the R42H genome contains 77 ORFs, of which 25 (32.47%) were annotated as functional genes. These functional genes were classified into structural proteins, DNA metabolism, DNA assembly, host lysis, and accessory metabolism-related genes, with the remainder being putative protein-related genes (Figure 6). Furthermore, no tRNA-encoding genes, virulence factors, or drug resistance-related genes were detected in this bacteriophage strain.

[0087] Of the 25 known functional genes in bacteriophage R42H, 6 are related to DNA metabolism, such as ORF3 endonuclease (HNH endonuclease), ORF18 homing endonuclease, ORF63 exonuclease, ORF53 DNA helicase (DnaB-like DNA helicase), and ORF76 tRNA pseudouridine synthase D. 15 genes are related to structure and assembly, such as the ORF6 terminase large subunit (which inserts a single viral genome into the viral procapsid through a process called "packaging"; it consists of a small subunit and a large subunit), ORF5 DNA packaging protein, ORF12 portal protein (through which bacteriophages inject DNA into host cells), ORF13 head protein, and ORF25... Tail fiber protein, ORF30 tail length tape measure protein, ORF36 baseplate wedge subunit, and ORF28 tail assembly chaperone were identified. Two recombination proteins (ORF40 and ORF41) associated with integrated functional genes were identified; genes containing two key cleavage proteins, endolysin and N-acetylmuramoyl-L-alanine amidase, correspond to ORF66 and ORF67 of bacteriophage R42H, respectively.

[0088] Example 9: Genetic and phylogenetic analysis of bacteriophages

[0089] 1. Method

[0090] The DNA packaging mechanism and sequence ends of the phage genome were identified using the PhageTerm online site on the Galaxy server (http: / / galaxy.pasteur.fr). The phage genome was analyzed using the GeneMarkS online software (http: / / exon.gatech.edu / GeneMark / index.html) and the ORF Finder online software (https: / / www.ncbi.nlm.nih.gov / orffinder / ) for Open Reading Frame (ORF) prediction. ORFs were determined using the BLASTP search algorithm on the Non-Redundant (NR) protein database of the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov) with an E-value ≤ 10. -5 Each translated ORF was searched and compared using a threshold, and ORFs were annotated and their functions predicted based on the protein functions encoded by the obtained homologous genes. The tRNA-coding sequences in the genome were identified using the tRNAscan-SE v.2.0 online software (http: / / lowelab.ucsc.edu / tRNAscan-SE / ). Phage virulence genes were detected in the Virulence Factor Database (VFDB, http: / / www.mgc.ac.cn / VFs / main.html). Antibiotic resistance genes were detected in the Comprehensive Antibiotic Resistance Database (CARD, https: / / card.mcmaster.ca / analyze / rgi). Phage AI, an online platform based on the whole phage genome, was used to predict the lifestyle of phages.

[0091] BLASTn (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to identify the phages most similar to the bacteriophage genome. Their sequences were downloaded from Genbank (https: / / www.ncbi.nlm.nih.gov) and their genomes were aligned using Clinker. The online website VICTOR (https: / / ggdc.dsmz.de / victor.php) was built based on viral classification and phylogenetic trees to analyze phage relationships. Furthermore, VIRIDIC (Virus Intergenomic Distance Caculator) was used to compare the similarity between phage genomes.

[0092] 2. Results

[0093] Based on NCBI BLASTN analysis, 10 bacteriophages showed high sequence identity with R42H (Table 1). Among them, bacteriophage V039C (NCBI sequence number MN_956515.1), isolated from various shrimp farms in Zhangpu, Fujian, showed the highest similarity to R42H, with a sequence identity of 87.61%, but the coverage was only 60%. VICTOR phylogenetic analysis (Figure 7, A) further confirmed that bacteriophage R42H and bacteriophage V039C clustered together, indicating a close phylogenetic relationship. R42H also clustered with bacteriophages such as PGA, PGB, and BT-1011 in a high-support branch, indicating that these bacteriophages are most closely related to R42H in terms of whole-genome evolution. Bacteriophage V039C and R42H also showed high similarity in their isolation origin, both being isolated from shrimp farms and sharing Vibrio alginolyticus as their host bacterium. Genome similarity analysis using VIRDIC showed (Figure 7, B) that phage R42H shared the highest genome similarity (56.8%) with phage V039C, perfectly consistent with the close phylogenetic relationship conclusion. However, since their ANIs were less than 70%, phage R42H was defined as a new genus. Phage R42H was named *Vibrio alginolyticusphage vB_ValM_R42H* and deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC No: 67434-B1, located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China.

[0094] The whole genome alignment of bacteriophage R42H (C in Figure 7) shows that the genome of bacteriophage R42H has certain similarities with the genomes of VO39C, PGA, and PGB. The genomes of the three bacteriophages can be divided into structural protein-related, DNA metabolism, DNA assembly, host lysis, and auxiliary metabolism-related genes according to their functions.

[0095] Table 1. Basic information on similar phages to phage R42H

[0096] .

[0097] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. Vibrio alginolyticusphage vB_ValM_R42H, characterized in that, The depositary institution is Guangdong Provincial Center for Microbial Culture Collection, and the accession number is GDMCC No: 67434-B1.

2. The use of the bacteriophage vB_ValM_R42H according to claim 1 in the preparation of a product for killing Vibrio alginolyticus.

3. The application according to claim 2, characterized in that, The product is a bactericide or biological control agent.

4. The application according to claim 2, characterized in that, The product is a disinfectant or water purifier used in aquaculture environments.

5. The application according to claim 2, characterized in that, The product is an animal feed additive or feed premix.

6. A composition for killing Vibrio alginolyticus, characterized in that, It contains an effective amount of the bacteriophage vB_ValM_R42H as described in claim 1 and a pharmaceutically or aquaculture-acceptable carrier.

7. The composition according to claim 6, characterized in that, The dosage form of the composition is a liquid, wettable powder, granules, lyophilized powder for injection, or microcapsule.

8. A method for killing Vibrio alginolyticus for non-disease treatment purposes, characterized in that, This includes applying an effective amount of the bacteriophage as described in claim 1 to the Vibrio alginolyticus.

9. The method according to claim 8, characterized in that, The application process is carried out by splashing or spraying water.

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