Lytic bacteriophage vBVcaPR24D and application thereof

Abstract

The invention discloses a lytic bacteriophage vBVcaPR24D and an application of the lytic bacteriophage vBVcaPR24D. The collection number of the lytic bacteriophage vBVcaPR24D is GDMCC (Graphical Digital Microbial Culture Collection Center) No: 65166-B1. The lytic bacteriophage vBVcaPR24D provided by the invention has a high specificity and a remarkable killing effect on vibrio cantoniensis V.campbelli LMG 11216T, and can be used for killing vibrio cantoniensis V.campbelli LMG 11216T. Besides, virulence factor genes and drug-resistant genes are not detected in the genome of the bacteriophage, so that the bacteriophage is high in safety and environment-friendly, and can be used as a bactericide for aquatic vibrio candersonii diseases.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, specifically to a lytic bacteriophage vB_VcaP_R24D and its applications. Background Technology

[0002] Vibrio are halophilic, Gram-negative bacteria widely distributed in estuarine and coastal ecosystems, and are considered opportunistic pathogens. Common pathogenic Vibrio species include *Vibrio cholera*, *V. alginolyticus*, *V. parahaemolyticus*, *V. campbellii*, and *V. harvestyi*. Increasingly, literature reports various aquatic animal diseases caused by Vibrio. For example, *V. parahaemolyticus*-induced lethal hepatopancreatic creatic necrosis disease (SHAD) and acute hepatopancreatic creatic necrosis disease (PHAD) are serious aquaculture diseases affecting shrimp, causing significant economic losses globally. *V. alginolyticus*-induced Zoea II syndrome also leads to molting damage and mass mortality in Litopenaeus vannamei. Currently, there is relatively little research on the pathogenicity of Vibrio canis, but existing literature indicates that it can cause aquatic diseases such as luminescence disease and acute hepatopancreatic necrosis. Dong et al. isolated a strain carrying pir from a Litopenaeus vannamei shrimp farm. vp The gene-derived *Vibrio kaneri* (20130629003S01) caused typical AHPND symptoms in Litopenaeus vannamei shrimp immersed in its suspension to appear within 12 hours, with a cumulative mortality rate reaching 100% within 36 hours, demonstrating strong pathogenicity. Haldar et al. isolated 26 strains of *Vibrio kaneri* from diseased shrimp. Infection experiments using Artemia as a model showed that 8 strains of *Vibrio kaneri* were highly pathogenic, and 3 strains were moderately pathogenic. Moreover, the pathogenic strains could colonize the intestines of Artemia after 24 hours. Therefore, the pathogenicity and pathogenic potential of *Vibrio kaneri* should not be underestimated and should be given due attention.

[0003] However, the overuse of antibiotics has led to serious problems affecting environmental health, ecological stability, public health, and food safety, making the search for green and efficient alternatives to antibiotics an urgent priority. Bacteriophages, as natural antibacterial agents, can efficiently and specifically lyse the host, are environmentally and microbial-friendly, and have shown great potential in treating Vibrio diseases in aquaculture. Currently, many studies have isolated Vibrio phages and assessed their potential for treating Vibrio diseases in aquaculture by measuring their biological and omics characteristics. The lytic activity, bactericidal efficiency, and environmental stability of phages are all important criteria for evaluating their application potential and for developing phage cocktails. In aquaculture environments, the absolute lytic activity of phages is a crucial prerequisite for their application. It not only efficiently kills pathogens but also prevents horizontal gene transfer and the spread of drug resistance genes because absolutely lytic phages do not enter a lysogenic state, thus reducing the occurrence of bacterial resistance. The bactericidal effect of bacteriophages is also an important criterion for evaluating their application potential. Stalin et al. isolated a strain of Vibrio parahaemolyticus and its bacteriophage from a shrimp farming pond in India. The one-step growth curve and bactericidal curve of the bacteriophage both showed a high lytic ability against the host Vibrio parahaemolyticus (outbreak rate of 2.9 × 10⁻⁶). 8 PFU / min (OD value of the experimental group with added phage was consistently below 0.2). In the infection experiment, the survival rate of *Litopenaeus monodon* in the phage-treated group was significantly improved. Furthermore, the environmental stability of phages is an important criterion for evaluating their application in production, sales, and real-world environments. Cai et al. isolated a strain of *Vibrio alginolyticus* phage, vB_Va_Val-yong3. By evaluating its environmental stability, including thermal stability, pH tolerance, and UV tolerance, they demonstrated that vB_Va_Val-yong3 is chloroform-resistant and relatively UV-resistant. After stabilizing within the range of 4-50℃ and pH 5-12, a phage treatment experiment was conducted on *Portunus trituberculatus* larvae using the immersion method. Treatment with vB_Va_Valyong3 increased the larval survival rate from 33.33% to 51.67%.

[0004] In conclusion, bacteriophages are considered a potential antibiotic alternative due to their high host specificity and low environmental impact. They can be used to control or prevent bacterial infections, reduce antibiotic use, thereby reducing the development of bacterial resistance, providing a new and environmentally friendly disease control strategy for aquaculture, and contributing to the protection of the ecological environment and the improvement of food safety. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a lytic bacteriophage vB_VcaP_R24D and its application in the prevention and control of Vibrio diseases in aquaculture environments.

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

[0007] The first objective of this invention is to provide a lytic bacteriophage vB_VcaP_R24D, with accession number GDMCC No:65166-B1.

[0008] This invention uses Vibrio campbellii type strain V. campbellii LMG 11216 T Using this as a host, two highly lytic bacteriophages, vB_VcaP_R24D and vB_VcaP_R25D, were isolated from wastewater at the Xiamen Seafood Market. Morphological observation, whole-genome sequencing, and phylogenetic analysis determined the morphology and taxonomic position of these bacteriophages. vB_VcaP_R24D was named *Vibrio campbellii phage* vB_VcaP_R24D and deposited on September 20, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCC No: 65166-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 a phage composition comprising the lytic phage vB_VcaP_R24D.

[0010] A third object of the present invention is to provide the use of the above-described lytic bacteriophage vB_VcaP_R24D or bacteriophage composition in the prevention and / or killing of Vibrio kanehirae.

[0011] Preferably, the Vibrio campbellii is V. campbellii LMG 11216. T .

[0012] A fourth object of the present invention is to provide the use of the above-described lytic bacteriophage vB_VcaP_R24D or bacteriophage composition in the preparation of products for controlling Vibrio infection in aquaculture.

[0013] Preferably, the products include pharmaceuticals, disinfectants, water purifiers, feed or feed additives.

[0014] Preferably, the Vibrio infection includes Vibrio campbellii infection, wherein Vibrio campbellii is V. campbellii LMG 11216. T .

[0015] A fifth object of the present invention is to provide a product for controlling Vibrio infection in aquaculture, which contains the above-mentioned lytic bacteriophage vB_VcaP_R24D or a bacteriophage composition.

[0016] A sixth objective of this invention is to provide a method for in vitro prevention and control of harmful infections caused by Vibrio campbellii. This method utilizes the lytic activity of the aforementioned lysing bacteriophage vB_VcaP_R24D or a bacteriophage composition to kill Vibrio campbellii in vitro. Further, the Vibrio campbellii is V. campbellii LMG 11216. T .

[0017] This invention uses Vibrio campbellii LMG 11216 T Two bacteriophages, vB_VcaP_R24D and vB_VcaP_R25D, were isolated from aquaculture wastewater at the Xiamen seafood market. Both phages exhibited rapid plaque formation with clear centers containing only a few undystomped resistant Vibrio bacteria, indicating strong lytic activity against the host. Genomic analysis revealed that both R24D and R25D contain genes encoding lysozyme (ORF 37 in R24D; ORF 35 in R25D) and cell wall hydrolases (ORF 48 in R24D; ORF 45 in R25D), enabling them to effectively disrupt Vibrio cell wall structure and promote infection and lysis. Furthermore, neither phage contained lysogen-related genes, meaning they did not enter a lysogenic state during host infection. Their lysis-related genes effectively lysed the host, reducing the risk of horizontal gene transfer. Phage AI software predictions also confirmed that both phage strains possessed 100% lytic potential. Absolute lytic activity of phages is a prerequisite for their application in disease treatment. When applied to phage therapy, highly lytic phages can rapidly and effectively kill host bacteria, control pathogen numbers, thereby limiting infection spread and accelerating pathogen clearance. Furthermore, compared to lysogenic phages, direct bacterial lysis reduces the possibility of drug resistance mutations. Through efficient pathogen control and reduced drug resistance risk, the treatment efficiency in aquatic environments is significantly improved.

[0018] One-step growth curves revealed that both phages followed a short latency period and low burst size, belonging to the "fast-low strategy" within the r strategy. These phages possess a fast life cycle and low reproductive output, rapidly lysing host cells during infection. Due to their small burst size, they have strong local infectivity and are not easily spread, limiting phage mutations and bacterial resistance. Phages with this "fast-low strategy" have great application potential in rapidly controlling pathogens without triggering excessive immune responses or disrupting the stability of the existing microbial community. Phages R24D and R25D have relatively small genomes (R24D is 44,615 bp, and R25D is 43,710 bp), containing genes encoding lysozyme and cell wall hydrolases, as well as abundant tail protein structures. Studies have shown that highly specific tail fibrin can rapidly recognize and adsorb onto the surface receptors of host bacteria, while perforin or tail needle-like structures rapidly penetrate the cell wall to inject phage genes. The diversity and functionalization of tail proteins also ensure the precision of DNA injection, and the small genome size shortens the time for phage DNA injection into host cells as well as the time for progeny phage DNA production and assembly. Efficient host recognition, rapid cell penetration, and synergistic effects with lysis-associated proteins ultimately accelerate the lysis process. This short latency and small lysis rate strategy allows phages to rapidly lyse pathogenic bacteria in a short period of time, showing strong application potential in aquatic environments with high Vibrio disease incidence, especially high Vibrio density, where they can quickly control pathogen numbers and limit disease spread.

[0019] Genome-wide comparative analysis suggests that three unique functional genes in R24D (ORF4, OFRF25, and ORF40) may play a crucial role in its higher burst rates. ORF4, annotated as a SAM-dependent methyltransferase, is involved in DNA modification processes, particularly methylation, helping phages resist the host cell's restriction-modification system. By protecting its genome from host degradation stress, R24D more easily maintains higher levels of phage production. ORF25, annotated as phosphate valonate kinase, is involved in the isoprene biosynthesis pathway and participates in the generation of phage envelope components and other molecular precursors. By improving the efficiency of lipid and small molecule synthesis, R24D may optimize the assembly efficiency of its structural proteins and the formation of phage particles, thereby further increasing burst rates. This metabolic advantage may allow R24D to produce more progeny phages in a shorter time. In summary, the unique genomic features of R24D, especially the presence of methyltransferase and mevalonate phosphate kinase, promote higher progeny phage yields by enhancing genomic protection and metabolic efficiency, resulting in a significantly higher burst size than R25D.

[0020] Genetic safety assessment of Vibrio phages is a necessary prerequisite for their application in treating Vibrio diseases in aquaculture. Genetic safety not only affects the effectiveness of phage therapy but also relates to the health of the environment and farmed organisms. Vibrio phages used for aquaculture treatment must ensure that their genomes do not contain any toxin genes or pathogenic factors that could be harmful to aquatic animals, preventing serious toxicity to farmed animals after toxins are released into the aquatic environment. Furthermore, the phage genome should not carry antibiotic resistance genes (ARGs) to prevent the enhancement of antibiotic resistance in pathogenic bacteria during treatment. Simultaneously, the phage should not carry integrative genetic elements such as integrase genes or transposons. These elements facilitate the integration of phage genes into the host bacterial genome, increasing the likelihood of genetic variation in the host bacteria and promoting horizontal gene transfer, posing a potential risk of pathogen evolution. Genomic analysis showed that no virulence factors, antibiotic resistance-related genes, or integrase genes were detected in the genomes of phages R24D and R25D, indicating that phages have good application prospects in terms of genetic safety.

[0021] The lysis curves demonstrate the significant potential of bacteriophages in treating Vibrio diseases in aquaculture. Both bacteriophage strains exhibit strong lytic activity against host Vibrio, making them suitable for phage therapy. They can effectively control the abundance of host Vibrio in the early stages, thus controlling Vibrio diseases. The phages showed the strongest lytic effect on host Vibrio growth when co-cultured in a host-phage mixed system for 6 hours, with lysis efficiency reaching up to 90% under different MOI conditions. Furthermore, due to the short latency of both bacteriophage strains, they significantly inhibited the growth of host Vibrio during the logarithmic growth phase, reducing the OD of host Vibrio. 600 When controlled within 0.4 g, the efficient lysis effect gives it a significant advantage in preventing Vibrio diseases. In laboratory studies using pure strains, although phage-resistant Vibrio can regrow in the later stages of culture, in practical applications, other bacterial groups or added probiotics can replace the resistant bacteria as the dominant group. Therefore, the combined application of phages and probiotics is also worthy of attention. Furthermore, R24D and R25D did not show a significant synergistic effect in the mixed system, possibly because the two phages have highly similar biological characteristics, leading to competition in the mixed culture system. This experimental result will also guide subsequent research on phage cocktails for Vibrio control. When developing and designing phage cocktails, the interactions and synergistic dynamics between the constituent phages should be considered to minimize the impact of competition between phages on the host lysis efficiency. For the same host, phage cocktails containing phages with different life histories or life strategies can achieve more comprehensive host control effects. For example, combining a phage with a rapid lysis cycle with a phage with a long latency period but strong diffusion ability can not only achieve rapid infection control but also provide sustained control effects over a long period of time.

[0022] Temperature stability and pH tolerance results showed that both bacteriophage strains exhibited good stability in aquaculture environments. Both strains maintained high activity (survival rate >90%) below 35℃, only becoming completely inactive above 55℃-65℃. Both strains showed activity within a pH range of 3-11, demonstrating a wide pH tolerance range, with an optimal pH of 7-8. Before applying bacteriophages to aquaculture environments for treatment, the preparation process, storage conditions, transportation process, and actual environmental conditions should be considered. Excessively high temperatures or pH fluctuations may affect bacteriophage activity and therapeutic efficacy. The suitable water temperature range for aquaculture is 24℃-30℃, and the pH range is 7.5-8.5. Therefore, both bacteriophage strains are fully adaptable to aquaculture environments and can realize their therapeutic potential in actual aquaculture practices. Furthermore, the environmental stability results provide a reference for the production, preparation, sales, transportation, storage, and optimal use conditions of the bacteriophages.

[0023] The beneficial effects of this invention are as follows:

[0024] The lytic bacteriophage vB_VcaP_R24D in this invention is a novel lytic short-tailed bacteriophage, effective against the pathogenic Vibrio campbellii LMG 11216. T This bacteriophage exhibits high specificity, effectively killing and controlling related Vibrio diseases. It possesses 100% lysis potential, follows a short incubation period and low outbreak rate strategy, and can rapidly lyse pathogenic bacteria in a short time. Furthermore, no virulence factor genes or drug resistance genes were detected in this bacteriophage, indicating that it is non-toxic, highly safe, and environmentally friendly. This demonstrates that the lytic bacteriophage vB_VcaP_R24D can be used as a bactericide against Vibrio alginolyticus diseases in aquaculture.

[0025] The Vibrio campbellii phage vB_VcaP_R24D of this invention was deposited on September 20, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), with accession number GDMCC No:65166-B1, located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China. Attached Figure Description

[0026] Figure 1 Morphological characteristics of bacteriophages R24D and R25D. (A) Plaques of bacteriophages R24D and R25D; (B) Electron micrographs of bacteriophages R24D and R25D.

[0027] Figure 2 One-step growth curves for bacteriophages R24D and R25D. Each data point is the mean ± standard error of three biological parallels.

[0028] Figure 3 This is the complete genome map of bacteriophages R24D and R25D. Differentially expressed genes are indicated within the dashed boxes.

[0029] Figure 4 Phage phylogenetic analysis and whole-genome alignment. (A) Whole-genome phylogenetic tree of R24D and R25D; (B) VIRIDIC heatmap of R24D and R25D; (C) Whole-genome alignment of R24D, R25D, and VEN.

[0030] Figure 5 The bactericidal effect of bacteriophages is shown in the following figures: (A) lysis curve of R24D; (B) lysis curve of R25D; (C) lysis curve of a 1:1 mixture of R24D and R25D; (D) lysis efficiency of different systems co-cultured for 6 hours. Each data point represents the mean ± standard error of 6 biological parallels.

[0031] Figure 6Environmental stability of bacteriophages. (A) Survival rates of bacteriophages R24D and R25D at temperatures ranging from 4°C to 75°C; (B) Survival rates of bacteriophages R24D and R25D at pH values ​​ranging from 2 to 13. Each data point represents the mean ± standard error of three biological parallels. Detailed Implementation

[0032] The following embodiments are further illustrations of the present invention, but not limitations thereof. Unless otherwise specified, the methods and techniques used are conventional.

[0033] Example 1: Isolation and purification of bacteriophages

[0034] This study used Vibrio campbellii LMG 11216 as a control group. T Phage isolation was performed for the host Vibrio species. This strain, a type strain, was purchased from the Belgian Microbial Collection Center (BCCM). *V. campbellii* LMG 11216 T They are usually cultured in 2216E liquid medium (10M tryptone, 2M yeast extract, artificial seawater at pH 7.5) and cultured on a shaker (160 rpm·min). -1 Water samples were collected from wastewater pipes of the Xiamen No. 8 Seafood Market and Xiamen Merchants Seafood Market in July 2023 for phage isolation. The water samples were filtered through a 0.22 μm polycarbonate membrane (Polycarbonate, Millipore, USA) and stored in a laboratory refrigerator at 4°C. Before the isolation experiment, the filtered water samples were added to *V. campbellii* LMG 11216 spores in the early logarithmic growth stage. T In bacterial culture, shake incubation (160 rpm·min) -1 After 24 hours at 28℃, the solution was filtered for sterilization to enrich potential phages. The filtrate was serially diluted with SM buffer (5.844g NaCl, 2.4647g MgSO4·7H2O, 6.057g Tris, 0.1g Gelatin, pH 7.5, 1L distilled water, autoclaved at 110℃ for 30 min), and phages were isolated using a double-layer agar plate method. Plaque formation was observed. Individual plaques were picked and purified for at least five rounds until the morphology and size of the plaques on the plates were consistent. Plaques were then stored in 1mL of SM buffer for further use, yielding a purified phage stock solution, which was stored at 4℃ protected from light.

[0035] Example 2: Enrichment of bacteriophages

[0036] 1 mL of purified phage was inoculated into 50 mL of host bacterial culture in the early logarithmic growth phase. Co-culture was continued until the culture medium became clear or showed signs of lysis such as flocculent formation. Cell debris was removed by centrifugation (10,000 × g, 4 °C, 15 min), and the supernatant was filtered through a 0.22 μm filter and inoculated into 200 mL of host bacterial culture in the early logarithmic growth phase. The above steps were repeated to recover the filtrate, gradually expanding the culture system to 1 L. A final concentration of 1 μg·mL⁻¹ was added to each 1 L host-phage co-culture medium. -1 DNase I and RNase A were digested at room temperature for 1 hour to degrade free DNA and RNA in the phage suspension. NaCl was then added to a final concentration of 1 mol / L, and after complete dissolution, the suspension was incubated at 4°C (or on ice) for approximately 1 hour to promote phage detachment from fragments and 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. 10% (wt / vol) of polyethylene glycol 8000 (PEG 8000) was added to the filtrate, and the suspension was incubated at 4°C in the dark for 1–3 days. After significant flocculent sedimentation, the suspension was centrifuged (12,000 × g, 4°C, 15 min), the supernatant was discarded, and the precipitate was resuspended in 6 mL of SM buffer. Cesium chloride (CsCl) density gradient centrifugation was performed. 2 mL of CsCl solutions with densities of 1.7, 1.5, and 1.3 g / mL were slowly added sequentially along the centrifuge tube wall. Finally, phage concentrate was added to the upper layer, balanced with SM buffer, and ultracentrifuged (200,000 × g, 4 °C, 24 h). The concentrated blue or blue-white phage band in the middle was aspirated, centrifuged (5,000 × g, 4 °C, 5 min) using a 30 kDa ultrafiltration tube (Millipore, USA), dialyzed to remove CsCl, and then stored in SM buffer at 4 °C in the dark.

[0037] Example 3: Morphological observation of bacteriophages

[0038] Morphage morphology was observed under electron microscopy using phosphotungstic acid negative staining. 10 μL of phage with an infectivity level of 10... 10 PFU·mL -1 The concentrated phage solution was dropped onto a 200-mesh copper grid coated with a carbon film. After adsorption in the dark for 10-30 minutes, it was negatively stained with 1% phosphotungstic acid for about 20 minutes and dried for more than 30 minutes. The sample was observed using a JEM 2100 transmission electron microscope (Japan Electron Optics Laboratory Co., Tokyo, Japan) at 120 kV. Images were acquired using a GATAN INC CCD image transmission system.

[0039] In this study, *Vibrio campbellii* LMG 11216 was used. T Two bacteriophages, vB_VcaP_R24D and vB_VcaP_R25D, were isolated from seafood aquaculture wastewater samples collected from the Xiashang Seafood Market (118.10°E, 24.55°N) and the Bashi Seafood Market (118.07°E, 24.46°N), respectively. Using the double-layer plate method, rapid plaque formation was observed for both bacteriophages. Clear, translucent, circular plaques approximately 1 mm in diameter were visible to the naked eye after about 5 hours of incubation, and the plaque diameter gradually increased with prolonged incubation time. Figure 1 A). The expansion rate of R24D plaque diameter within 12 h is approximately 2.1 μm·h. -1 The final diameter is 4-5 mm, while the R25D plaques are slightly smaller, with an expansion rate of approximately 0.67 μm·h. -1 The final diameter was 3 mm. The plaques formed by the two phages were clear and transparent, and almost all Vibrio bacteria in the plaque area were lysed, reflecting the extremely strong lytic activity of the two phages. In addition, both plaques had a semi-transparent halo around their periphery. Figure 1 A), is often thought to be related to polysaccaride depolymerase within the bacteriophage. TEM morphological results show that bacteriophages R24D and R25D are both short-tailed bacteriophages (A). Figure 1 B) consists of an icosahedral head (R24D: head length 60±2.87nm, head width 69±1.23nm; R25D: head length 58±2.67nm, head width 57±2.16nm) and a short, non-contractable tail (rich in tail filament structure).

[0040] Example 4: Chloroform susceptibility assay of bacteriophages

[0041] Chloroform susceptibility assays can assess the presence of lipid-related compounds in bacteriophage capsids. 1 mL of chloroform with an infectivity of 10... 8 PFU·mL -1 Phage solutions were mixed with chloroform to achieve final concentrations of 0%, 2%, and 20%, respectively. The mixtures were shaken for 30 seconds, incubated at room temperature in the dark for 30 minutes, and then centrifuged at low speed (5,000 × g, 4 °C, 5 min) for phase separation. 2 μL of the supernatant was dropped onto V. campbellii LMG 11216. T After cooling on agar plates, the plates were incubated at 30°C. Plaques were observed after formation. Each treatment was performed in triplicate, with SM buffer used as a negative control instead of phage fluid.

[0042] The chloroform sensitivity test results 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 the two phage strains R24D and R25D were not sensitive to chloroform, did not contain lipid substances in their capsids, and were not encapsulated by a lipid membrane.

[0043] Example 5: One-step growth curve of bacteriophage

[0044] One-step growth curves can be used to study the infection, replication, and lysis kinetics of bacteriophages. The host bacterium *V. campbellii* LMG 11216 was inoculated at a volume ratio of 1:50. T In 2216E liquid medium, shake incubate (160 rpm·min). -1 (30℃) to the early logarithmic growth stage (OD) 600 =0.1-0.2). A certain amount of phage was added to the host bacterial culture in the early stage of logarithmic growth to make its multiplicity of infection (MOI) 0.01-0.05. After the phage-host mixture was allowed to adsorb in the dark for 5 min, it was centrifuged (8,000×g, 4℃, 4 min), the supernatant was discarded, and 1 mL of 2216E liquid medium was taken to resuspend the precipitate. The centrifugation operation was repeated twice to fully remove free phages that had not been adsorbed to the host cells. Finally, 1 mL of the resuspended solution was inoculated into 50 mL of 2216E liquid medium and cultured on a shaker (160 rpm·min). -1 Phage infectivity was measured every 10 minutes at 30°C for one hour. Each treatment was repeated in triplicate.

[0045] One-step growth curves can be used to investigate the latency and lysis rate of bacteriophages, i.e., the time required for a bacteriophage to replicate, invade, assemble, and release progeny phages, and the average number of progeny phages released from each infected host cell. The one-step growth curve results showed that the latency of both bacteriophage strains was less than 10 minutes, indicating that both bacteriophages rapidly replicated and assembled within the host cell and quickly lysed the host cell. Figure 2 During phage release, R24D (32±2 PFU·cell) -1 The progeny phage particle release was consistently greater than that of R25D (17±1 PFU·cell). -1 (P<0.01), its lysis rate was nearly twice that of R25D. In summary, both bacteriophages follow a life strategy of short latency and small outbreak, and the lysis rate of R24D is higher than that of R25D.

[0046] Example 6: Host lysis profile of bacteriophages

[0047] To determine the host lysis profiles of bacteriophages R24D and R25D and compare their differences in host range, this study selected 26 Vibrio strains for experiments, including 4 *Vibrio cannii* strains, 4 *Vibrio harveyi* strains, 2 *Vibrio erwinis* strains, 4 *Vibrio alginolyticus* strains, 9 *Vibrio parahaemolyticus* strains, 1 *Vibrio anguillarum* strain, 1 *Vibrio fluvialis* strain, and 1 *Vibrio brasiliensis* strain isolated from the hepatopancreas of farmed shrimp. This study used plaque titration to determine the host range of bacteriophages. 1 mL of Vibrio culture medium cultured to the early logarithmic growth stage was mixed with 5 mL of melted 2216E semi-solid medium and poured onto a 2216E solid culture plate to cool and solidify. 2 μL of the bacteriophage liquid (approximately 10% infectious) was then taken. 8 PFU·mL -1 The plaques were dropped onto a cooled and solidified Vibrio agar plate, allowed to cool completely, and then incubated at 30°C to observe plaque formation. Each treatment was performed in triplicate, using Vibrio campbellii LMG 11216 as the host bacterium. T The test strain was used as a positive control, and SM buffer was used as a negative control.

[0048] The host range tests used Vibrio species commonly found in aquaculture environments, including Vibrio campbellii, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio erwinis, Vibrio brasiliensis, and Vibrio fluvibrio. These were isolated from various habitats, including aquaculture water and aquatic animal tissues. The host range results showed that bacteriophages R24D and R25D exhibited narrow host lysis ranges, almost exclusively infecting the isolated host Vibrio campbellii LMG 11216 among the tested Vibrio species. T This indicates that the two bacteriophage strains exhibited strong host specificity (Table 1). However, this study cannot rule out the possibility that the two bacteriophage strains could infect other untested hosts.

[0049] Table 1. Host range of bacteriophages R24D and R25D

[0050]

[0051]

[0052] Note: "+" indicates that it can infect, and "-" indicates that it cannot infect.

[0053] Example 7: Extraction and sequencing of bacteriophage DNA

[0054] DNA from the pure phage strain was extracted using the phenol-chloroform extraction method. 1.5 μL of 100 mg / mL solution was added to the concentrated and enriched phage suspension. -1 Proteinase K solution, 100 μL of 10% (wt / vol) SDS solution, 10 μL of 0.5 mol·L⁻¹-1 The EDTA solution was mixed and then incubated in a 55°C water bath for 3 hours. An equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, volume ratio) mixture was added to the digested phage suspension, mixed well, and centrifuged (12,000×g, 4°C, 5 min). The upper aqueous phase was transferred to a new centrifuge tube, and this step was repeated twice. Add an equal volume of chloroform-isoamyl alcohol mixture (24:1, volume ratio) to the collected upper aqueous phase solution, shake thoroughly for 30 s, centrifuge (12,000×g, 4℃, 5 min), collect the upper aqueous phase solution, mix with an equal volume of isopropanol liquid, incubate at -20℃ for at least 1 h, centrifuge again (12,000×g, 4℃, 5 min), collect the phage nucleic acid precipitate, wash twice with pre-cooled 70% ethanol solution, air dry, and then dissolve the nucleic acid precipitate in 100 μL of TE buffer (10 mmol·L⁻¹). -1 Tris-HCl, 1 mmol·L -1 EDTA (pH=8.0) was stored at -20℃. Phage whole-genome sequencing was performed by Shanghai Hanyu Biotechnology Co., Ltd. (China). The sequencing library was prepared by... Ultra TM DNA library was constructed using a DNA library preparation kit (NEB, USA) and sequenced using an Illumina HiSeq 4000 platform.

[0055] Example 8: Genomic analysis of bacteriophages

[0056] Raw data and genome assembly were analyzed using Trimmomatic v0.32 and Velvet v1.2.03 software. 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). Open reading frames (ORFs) were predicted using the GeneMarkS (http: / / exon.gatech.edu / GeneMark / index.html) online software and the ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) online software. ORFs were determined using the BLASTP search algorithm based 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. -5Each 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.

[0057] Both phages R24D and R25D possess linear double-stranded DNA with total lengths of 44,615 bp and 43,710 bp, respectively, and G+C contents of 43.69% and 43.74%, respectively. Using PhageTerm to identify phage ends and predict packaging sites, both phages R24D and R25D belong to the COS (Cohesive End Sites) packaging site group. R24D has a 357 bp direct terminal repeat sequence, while R25D has a 334 bp direct terminal repeat sequence. The ORFs of bacteriophages R24D and R25D were predicted using GenemarkS. R24D had 51 ORFs, all of which were on the positive strand except for the first one. BLAST alignment of the 51 predicted ORFs was performed to predict gene function (Table 2), with 24 (47.05%) functional genes annotated. R25D had 48 ORFs, all on the positive strand, with 22 (45.83%) functional genes annotated. Based on the annotated functions, the genes of the two bacteriophages 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 3 Furthermore, no tRNA, virulence factors, or drug resistance-related genes were detected in either phage strain.

[0058] Of the known functional genes in the two bacteriophage strains, approximately 26% are related to DNA metabolism, including genes related to RNA polymerase (T7-like RNA polymerase), DNA helicase (RecA-like domain of Escherichia colibacteriophage T7 Gp4Dhelicase), DNA polymerase I, DNA ligase, 5'-3' exonuclease, and endonuclease VII; approximately 50% of the genes are related to structure and assembly, such as the large and small subunits of terminase, and abundant structural proteins, such as head-tail connecting protein, head scaffolding protein, capsid and scaffold protein, tail tubular protein, tail protein, internal virion protein, and tail fibrin. Most of these genes are related to the structure of proteins in the phage tail, such as fiber protein. In terms of host lysis, both phages contain genes encoding cell wall hydrolases, corresponding to ORF 48 of R24D and ORF 45 of R25D, respectively. Both phage genomes also contain a 699bp gene encoding a protein containing a lysozyme domain (R24D ORF37, R25D ORF 35). The genomes of both phages also contain some accessory metabolic genes related to host metabolism, such as genes encoding acetyltransferase (GNAT) family and acetyl-CoA acetyltransferase related to acetyltransferases (R24D: ORF 29, ORF 43, ORF 44; R25D: ORF 27, ORF 40, ORF 41).Meanwhile, phage R25D possesses a unique functional gene predicted to be PrgI family protein (ORF 4), while R24D possesses three unique functional genes (ORF4, OFRF25, and ORF40), which are respectively labeled as SAM-dependent methyltransferase, phosphate homevalonate kinase, and holin.

[0059] Table 2. ORF prediction gene functions of phages R24D and R25D

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[0064] Example 9: Phylogenetic analysis and classification of bacteriophages

[0065] BLASTn (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was used to identify the bacteriophages most similar to the genome of the bacteriophage, whose sequences were downloaded from Genbank (https: / / www.ncbi.nlm.nih.gov) and aligned using Clinker (Gilchrist, 2021#105). The online website VICTOR (https: / / ggdc.dsmz.de / victor.php) was built based on viral classification and phylogenetic trees to analyze bacteriophage relationships. Furthermore, VIRIDIC (Virus Intergenomic Distance Caculator) was used to compare the similarity between bacteriophage genomes.

[0066] Based on NCBI BLASTN analysis, 11 phages showed high sequence identity with R24D and R25D (Table 3). Among them, the Vibrio alginolyticus phage VEN (NCBI accession number NC_047903.1) isolated from Greece showed the highest similarity with R24D and R25D, with sequence identity of 92.32% and 92.17%, respectively, and coverage of 85% and 88%, respectively (Table 3). VICTOR phylogenetic analysis further confirmed that R24D and R25D clustered with VEN, indicating a close phylogenetic relationship among these three phages. Figure 4A). All three bacteriophages were isolated from environments related to aquaculture and sales. The difference lies in the host of bacteriophages R24D and R25D: *Vibrio kanehirae*, while the host of bacteriophage VEN was *Vibrio alginolyticus*. Figure 4 A). Genomic similarity analysis using VIRDIC showed that R24D and R25D shared 80.4% and 82.9% similarity with VEN, respectively, and the similarity between the R24D and R25D genomes was 90.7%, indicating that R24D, R25D, and VEN belong to the genus Trungvirus in the family Autographiviridae, but are different species within the Trungvirus genus. Figure 4 B) is a novel virus belonging to the Trungvirus genus. vB_VcaP_R24D was named *Vibrio campbellii phage* vB_VcaP_R24D and deposited on September 20, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), accession number GDMCCNo:65166-B1. The depositary address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, 510070, China.

[0067] Comparative analysis of the whole genomes of bacteriophages R24D, R25D, and VEN showed that, based on function, the genomes of all three bacteriophages could be divided into genes related to structural proteins, DNA metabolism, DNA assembly, host lysis, and accessory metabolism. Furthermore, genes related to DNA metabolism and tail structural proteins in the three bacteriophages showed high homology (91.58%-99.45%). Figure 4 C). By comparing the whole genomes of three closely related bacteriophages—R24D, R25D, and VEN—high homology was found in RNA polymerase, DNA primase, DNA polymerase, 5'-3' exonuclease, and Recombination endonuclease VII, indicating that these three bacteriophages share the same DNA metabolic processes and pathways. Regarding tail structure, the head-tail connecting protein and tail tubular protein of the three bacteriophages also showed high homology. Furthermore, all three bacteriophages possessed genes related to cell wall hydrolase and lysozyme domain-containing protein, indicating that all three bacteriophages are virulent. However, the gene encoding Holin was only detected in the genomes of bacteriophages R24D and VEN, while the genome of R25D showed a deletion of this Holin-encoding gene at the corresponding position.

[0068] Table 3. NCBI blanstn alignment results of bacteriophage R24D and R25D.

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[0071] Example 10: Determination of the bactericidal efficiency of bacteriophages

[0072] To investigate the effects of two bacteriophage strains on the host Vibrio campbellii LMG 11216 T The lytic activity of the phage was investigated, and bactericidal experiments on the host were conducted under different MOI conditions. Vibrio campbellii LMG 11216 was inoculated into the host organism at a volume ratio of 1:50. T Cultured in 2216E liquid medium on a shaker (160 rpm·min). -1 (30℃) to the early logarithmic growth stage (OD) 600 After adding 0.1-0.2% of the sample, bacterial culture and phage of 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 the samples in each well was then monitored in real time at 600 nm using a multi-mode microplate reader (Synergy H1, Bio-Tek, USA). The systems were co-cultured for 24 h, with measurements taken every 30 min. The plate was shaken for 5 seconds before each measurement, and the detection temperature was 28 °C. Each treatment was performed in at least five replicates, with 2216E liquid medium and bacterial culture serving as negative and positive controls, respectively.

[0073] Phages R24D and R25D inhibit the host *Vibrio campbellii* LMG 11216 under different MOI conditions. T The bactericidal test results are as follows Figure 5 As shown. By adding bacteriophage R24D, R25D, or a mixture thereof to the host Vibrio culture medium in the exponential growth phase, bacteriophage R24D, R25D, and the mixture can all rapidly lyse the host. Furthermore, the higher the MOI, the stronger the lytic effect of the bacteriophage on the host. In the early stages of culture, it can effectively reduce the OD of the host Vibrio. 600 Keep within 0.4 ( Figure 5 (A, 5B, 5C). After 6 hours of co-culture, the lytic activity of bacteriophages R24D, R25D, and the bacteriophage mixture against the host Vibrio growth reached its peak. At this point, the bacteriophage bactericidal efficiency reached approximately 90% under all MOI conditions (MOI of 0.001-10). Figure 5(D) Further analysis showed that 6 hours after phage addition, the bactericidal efficiency of phage R24D was slightly higher at an MOI of 0.001-0.1 than at an MOI of 0.1-10, indicating that adding a small amount of phage R24D could quickly achieve a high bactericidal effect. Adding a mixture of the two phages to hosts in the exponential growth phase did not significantly differ in lysis efficiency from adding the phage alone, indicating that mixing two similar phages did not improve their bactericidal efficiency.

[0074] Example 11: Thermal stability and pH tolerance of bacteriophages

[0075] 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... 8 PFU·mL -1 Phage solutions were prepared and incubated at different temperature gradients (4℃, 15℃, 25℃, 35℃, 45℃, 55℃, 65℃, and 75℃) for 6 hours in the dark. After returning to room temperature, the infectivity of the phages was determined using the double-layer plate method. SM buffer solutions with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 were prepared. Phage solutions were incubated in SM buffer solutions of different pH values ​​for 6 hours in the dark at room temperature, and the infectivity of the phages was determined using the double-layer plate method. Each treatment was performed in triplicate.

[0076] The environmental stability results of bacteriophages R24D and R25D are as follows: Figure 6 As shown, bacteriophages R24D and R25D maintained high activity (survival rate >90%) within the temperature range of 4℃-35℃, and their activity only decreased significantly when the temperature reached 55℃. Figure 6 A) When the temperature reaches 65℃, bacteriophage R24D is completely inactivated, while bacteriophage R25D still retains some activity (survival rate >4.5%) at 65℃, until it is completely inactivated at 75℃. In summary, both bacteriophages R24D and R25D exhibit good stability within the temperature range of 4℃-45℃; however, the activity of bacteriophage R24D is more susceptible to high temperatures and is more sensitive to temperature changes. Meanwhile, the optimal pH range for both bacteriophages is 7-8, with a survival rate as high as 95.56%-111.1%. Both bacteriophages maintain high activity (survival rate >80%) within the pH range of 4-10; only when the pH is below 3 or above 11 do the bacteriophages completely lose their activity. Data analysis showed no significant difference in survival rates between the two bacteriophages within a pH range of 3-10. However, at pH 11, the survival rate of bacteriophage R25D was significantly higher than that of R24D (P<0.05), indicating that bacteriophage R25D has a stronger tolerance to alkaline environments. Figure 6 B).

[0077] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Lytic bacteriophage vB_VcaP_R24D, deposit number: GDMCC No: 65166-B1.

2. A phage composition comprising the lytic phage vB_VcaP_R24D according to claim 1.

3. Use of the lytic bacteriophage vB_VcaP_R24D according to claim 1 or the bacteriophage composition according to claim 2 in preventing and / or killing Vibrio cannelli.

4. Use of the lytic bacteriophage vB_VcaP_R24D according to claim 1 or the bacteriophage composition according to claim 2 in the preparation of a product for controlling Vibrio infection in aquaculture.

5. The use according to claim 4, characterized in that: The products include medicines, disinfectants, water purifiers, feed or feed additives.

6. The use according to claim 4, characterized in that: The Vibrio infection includes Vibrio cannerii infection.

7. The use according to claim 3 or 6, characterized in that: The Vibrio campbellii is V. campbellii LMG 11216 T 。 8. A product for controlling Vibrio infection in aquaculture, characterized in that: Containing the lytic phage vB_VcaP_R24D according to claim 1 or the phage composition according to claim 2.

9. A method for preventing and treating harmful infections caused by Vibrio cannellii in vitro, characterized in that: Vibrio canneryii is killed in vitro by using the lytic bacteriophage vB_VcaP_R24D described in claim 1 or the bacteriophage composition described in claim 2 to lyse Vibrio canneryii.

10. The method according to claim 9, characterized in that The Vibrio campbellii is V. campbellii LMG11216 T .

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