Lytic bacteriophage vBVcaPR25D and application thereof

Abstract

The invention discloses a lytic bacteriophage vBVcaPR25D and an application of the lytic bacteriophage vBVcaPR25D. The collection number of the lytic bacteriophage vBVcaPR25D is GDMCC (Graphical Digital Microbial Culture Collection Center) No: 65167-B1. The lytic bacteriophage vBVcaPR25D 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] The present invention relates to the technical field of microbial application, and in particular to a lytic bacteriophage vB_VcaP_R25D and an application thereof. Background Art

[0002] Vibrio is a type of halophilic Gram-negative bacteria that is widely distributed in estuaries and coastal ecosystems and is a conditional pathogen. Common pathogenic Vibrio include Vibrio cholera, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio campbellii and Vibrio harveyi. More and more literature reports on various aquatic animal diseases caused by Vibrio, such as Septic Hepatopancreatic creatic Necrosis Disease (SHAD) and Acute Hepatopancreatic necrosis Disease (PHAD) caused by Vibrio parahaemolyticus, which are aquaculture diseases that seriously affect shrimps and have caused huge economic losses worldwide; Zoea II syndrome caused by Vibrio alginolyticus also causes the molting of white shrimp to be damaged and cause a large number of deaths. At present, there are relatively few studies on the pathogenicity of Vibrio cannerii, but existing literature shows that it can cause aquatic diseases such as luminous disease and acute hepatopancreatic necrosis. Dong et al. isolated a strain carrying pir from a shrimp farm. vp The vannamei shrimp immersed in the suspension of Vibrio cannonii began to show typical AHPND symptoms within 12 hours, and the cumulative mortality rate reached 100% within 36 hours, which was highly pathogenic. Haldar et al. isolated 26 strains of Vibrio cannonii from the diseased shrimp. The infection experiment with Artemia as a model showed that 8 strains of Vibrio cannonii were highly pathogenic, 3 strains of Vibrio cannonii were moderately pathogenic, and the pathogenic strains could colonize in the Artemia intestine after 24 hours. Therefore, the pathogenicity and pathogenic potential of Vibrio cannonii should not be underestimated and should be taken seriously.

[0003] However, the abuse of antibiotics has brought serious problems such as environmental health, ecological stability, life health and food safety. It is urgent to find green and efficient prevention and control methods to replace antibiotics. As a natural antibacterial agent, bacteriophage can efficiently and specifically lyse the host, is friendly to the environment and microbial community, and shows great potential in the treatment of Vibrio diseases in aquaculture. At present, many studies have isolated Vibrio phages and evaluated the potential of phages to treat Vibrio diseases in aquaculture by measuring their biological and omics characteristics. The characteristics of phage lysis, bactericidal efficiency and environmental stability are important bases for evaluating the application potential of phages and the preparation of phage cocktails. In an aquaculture environment, the absolute lysis of phages is an important prerequisite for their application. It can not only efficiently kill pathogens, but also prevent the transfer of horizontal genes and the spread of resistance genes because the absolutely lytic phages will not enter the lysogenic state, thereby reducing the occurrence of bacterial resistance. The bactericidal effect of phages is also one of the important criteria for evaluating their application potential. Stalin et al. isolated a strain of Vibrio parahaemolyticus and its phage from a Penaeus monodon culture pond in India. The one-step growth curve and bactericidal curve of the phage showed a high lysis ability against the host Vibrio parahaemolyticus (burst volume of 2.9×10 8 PFU / min, the OD value of the experimental group with added phages was always lower than 0.2), and the survival rate of the phage-treated group in the infection experiment was significantly improved. In addition, the environmental stability of phages is an important criterion for evaluating their production, sales and actual environmental applications. Cai et al. isolated a strain of Vibrio alginolyticus phage vB_Va_Val-yong3. By evaluating its environmental stability such as thermal stability, pH tolerance, and UV tolerance, it was proved that vB_Va_Val-yong3 was resistant to chloroform and relatively resistant to UV rays. After being stable in the range of 4-50°C and pH 5-12, the phage treatment test was carried out on the larvae of the three-spotted swimming crab by immersion method. The vB_Va_Valyong3 treatment increased the survival rate of the larvae from 33.33% to 51.67%.

[0004] In summary, bacteriophages are considered as a potential alternative to antibiotics due to their high host specificity and low environmental impact. They can be used to control or prevent bacterial infections, reduce the use of antibiotics, and thus reduce the development of bacterial resistance, providing a new and environmentally friendly disease control strategy for the aquaculture industry, helping to protect the ecological environment and improve food safety. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a lytic bacteriophage vB_VcaP_R25D and its application in preventing and controlling Vibrio diseases in aquaculture environments.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The first object of the present invention is to provide a lytic bacteriophage vB_VcaP_R25D, whose deposit number is: GDMCC No: 65167-B1.

[0008] The present invention uses the model strain V. campbellii LMG 11216 T As the host, two strong lytic phages vB_VcaP_R24D and vB_VcaP_R25D were isolated from the wastewater of Xiamen Seafood Market. The morphology and taxonomic status of the phage were determined by morphological observation, whole genome sequencing and phylogenetic analysis, and vB_VcaP_R25D was named Vibrio campbellii phage vB_VcaP_R25D. It was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on September 20, 2024, with the deposit number GDMCC No: 65167-B1. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070.

[0009] The second object of the present invention is to provide a phage composition comprising the lytic phage vB_VcaP_R25D.

[0010] The third object of the present invention is to provide the use of the above-mentioned lytic phage vB_VcaP_R25D or phage composition in preventing and / or killing Vibrio cannelli.

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

[0012] The fourth object of the present invention is to provide the use of the above-mentioned lytic bacteriophage vB_VcaP_R25D or bacteriophage composition in the preparation of products for controlling Vibrio infection in aquaculture.

[0013] Preferably, the product includes medicines, disinfectants, water purifiers, feed or feed additives.

[0014] Preferably, the Vibrio infection includes Vibrio campbellii infection, and the Vibrio campbellii is V. campbelliiLMG 11216 T .

[0015] The 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_R25D or bacteriophage composition.

[0016] The sixth object of the present invention is to provide a method for preventing and treating harmful infections of Vibrio campbellii in vitro, wherein the method utilizes the lytic bacteriophage vB_VcaP_R25D or the bacteriophage composition to kill Vibrio campbellii in vitro. Furthermore, the Vibrio campbellii is V. campbellii LMG 11216 T .

[0017] The present invention uses Vibrio campbellii LMG 11216 T As the host, two phages, vB_VcaP_R24D and vB_VcaP_R25D, were isolated from the aquaculture wastewater of Xiamen Seafood Market. The two phages were rapidly plaqued, with the center of the plaques being transparent, and only a few resistant Vibrio that were not lysed were present, indicating that the phages had strong lysis properties on the host. Genomic analysis showed that both R24D and R25D contained genes encoding lysozyme (ORF 37 in R24D; ORF 35 in R25D) and cell wall hydrolases (ORF 48 in R24D; ORF 45 in R25D), which enabled the phages to effectively destroy the cell wall structure of Vibrio, thereby promoting the infection and lysis of Vibrio by the phages. In addition, both phages did not contain lysogeny-related genes, and the phages would not enter the lysogeny state during the infection of the host. Under the action of their lysis-related genes, they could effectively lyse the host, reducing the risk of horizontal gene transfer. The prediction results using Phage AI software also confirmed that the two phages have 100% lysis potential. The absolute lysis of phages is a prerequisite for their application in disease treatment. When used in phage therapy, highly lytic phages can quickly and effectively kill host bacteria, control the number of pathogens, thereby limiting the spread of infection and accelerating pathogen clearance. At the same time, compared with lysogenic phages, direct lysis of bacteria reduces the possibility of drug-resistant mutations. Through efficient pathogen control and reduced resistance risks, the treatment efficiency of aquatic environments has been significantly improved.

[0018] The one-step growth curve revealed that both phages followed a life strategy with a short incubation period and a small burst size, which belongs to the "Fast-Low Strategy" in the r strategy. This type of phage has a fast life cycle and low reproductive output. It will quickly lyse host cells during the infection process. Due to its small burst size, it has a strong local infection ability and is not easy to spread, which limits the phage mutation and the generation of bacterial resistance. This phage with a "fast-low strategy" has great application potential in the case of rapid control of pathogens without inducing excessive immune responses and destroying the stability of the original microbial community structure. The genomes of phages R24D and R25D are small (R24D is 44,615bp, R25D is 43,710bp), containing genes encoding lysozyme and cell wall hydrolases, as well as rich tail protein structures. Studies have shown that highly specific tail fiber proteins can quickly recognize and adsorb to the surface receptors of host bacteria, while perforin or tail needle structures quickly penetrate the cell wall and inject phage genes. The diversity and functionalization of tail proteins also ensure the accuracy of DNA injection, and the smaller genome size also shortens the time for phage DNA injection into host cells and the production and assembly of progeny phage DNA. Efficient host recognition, rapid cell penetration, and synergistic effects with lysis-related proteins ultimately accelerate the lysis process. This life strategy with a short incubation period and a small amount of lysis enables phages to quickly lyse pathogenic bacteria in a short period of time. It has strong application potential in aquatic environments where Vibrio diseases are prevalent, especially in areas with high Vibrio density, and can quickly control the number of pathogens and limit the spread of diseases.

[0019] According to the whole genome comparative analysis, the three unique functional genes (ORF4, OFRF25, ORF40) possessed by R24D may play an important role in its higher burst. ORF4 is annotated as SAM-dependent methyltransferase, which is involved in DNA modification, especially methylation, and helps phages resist the restriction-modification system of host cells. By protecting its genome from the degradation pressure of the host, R24D is more likely to maintain a higher level of phage production; ORF25 is annotated as phosphomevalonate kinase, which is related to the isoprenoid biosynthesis pathway and participates in the generation of phage envelope components and other molecular precursors. By improving the synthesis efficiency of lipids and small molecules, R24D may optimize the assembly efficiency of its structural proteins and the formation of phage particles, thereby further increasing the burst. This metabolic advantage may allow R24D to produce more progeny phages in a short period of time. In summary, the unique genomic features of R24D, especially the presence of methyltransferase and phosphomevalonate kinase, promoted higher progeny phage production by improving genome protection and metabolic efficiency, resulting in a significantly higher burst output than R25D.

[0020] Genetic safety assessment of Vibrio phages is a necessary prerequisite for their application in aquaculture to treat Vibrio diseases. Genetic safety not only affects the effectiveness of phage therapy, but also affects 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 can encode toxic effects on aquatic animals, so as to avoid serious toxicity to farmed animals after toxins are released into the aquatic environment. In addition, the phage genome should not carry antibiotic resistance genes (ARGs) to prevent the enhancement of antibiotic resistance of pathogenic bacteria during treatment. At the same time, phages should not carry integrative genetic elements, such as integrase genes and transposons, which help phage genes integrate into the host bacterial genome, thereby increasing the possibility of genetic variation in host bacteria and promoting horizontal gene transfer, bringing potential risks of pathogen evolution. Genomic analysis showed that no integrative genetic elements such as virulence factors, antibiotic resistance-related genes or integrases 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 show the important application potential of phages in the treatment of Vibrio diseases in aquaculture. The two phages have strong lysis properties against host Vibrio and are suitable for phage therapy. They can effectively control the abundance of host Vibrio in the early stage and prevent and control Vibrio diseases. When the host-phage mixed system is co-cultured for 6 hours, the phage has the strongest lysis effect on the growth of host Vibrio, and the lysis efficiency is as high as 90% under different MOI conditions. At the same time, due to the biological characteristics of the two phages with a short incubation period, they can significantly inhibit the growth of host Vibrio during the logarithmic growth period, and the OD of the host Vibrio can be reduced by 1.5%. 600 Controlled within 0.4, the efficient lysis effect gives it a clear advantage in preventing Vibrio diseases. In laboratory pure strain studies, although phage-resistant Vibrio can grow again in the later stage of culture, in actual application, other flora or added probiotics can replace resistant bacteria to become the dominant flora, so the combined application of phages and probiotics is also worthy of attention. In addition, R24D and R25D did not show obvious synergistic effects in the mixed system, which may be due to the highly similar biological characteristics of the two phages, forming a competitive relationship in the mixed culture system. The experimental results will also guide subsequent phage cocktail research on the application of mixed phage preparations to Vibrio prevention and control. When developing and designing phage cocktail preparations, the interactions and synergistic dynamics between the component phages must be taken into account to minimize the high efficiency of host lysis due to competition between phages. For the same host, phage cocktails made from phages with different life histories or life strategies can achieve a more comprehensive host prevention and control effect. For example, selecting a phage with a fast lysis cycle and combining it with a phage with a long incubation period but strong diffusion ability can not only achieve rapid infection control, but also provide sustained prevention and control effects over a long period of time.

[0022] The results of temperature stability and pH tolerance showed that the two phages have good stability in the aquaculture environment. Both phages can maintain high activity below 35°C (survival rate>90%) and are completely inactivated when the temperature exceeds 55°C-65°C. The two phages have certain activity in the pH range of 3-11 and a wide pH tolerance range, with the optimum pH being 7-8. Before the phages are used for treatment in the aquaculture environment, their preparation process, storage conditions, transportation process and environmental conditions during actual use should be considered. When the ambient temperature is too high or the pH changes, the phage activity and the effect of phage treatment may be affected. The water temperature range suitable for aquaculture is between 24°C-30°C and the pH is between 7.5-8.5. Therefore, the two phages can fully adapt to the aquaculture environment and exert the potential of phage treatment in the actual aquaculture process. In addition, the environmental stability results also provide a reference for the production, preparation, sales, transportation, storage and optimal environmental conditions of phages.

[0023] The present invention has the following beneficial effects:

[0024] The lytic bacteriophage vB_VcaP_R25D of the present invention is a new lytic short-tailed bacteriophage, which is effective against the pathogenic bacteria V. campbellii LMG 11216. T It is highly specific and can specifically and effectively kill and prevent related Vibrio diseases. The phage has 100% lysis potential, follows a life strategy of short incubation period and small burst, can quickly lyse pathogenic bacteria in a short period of time, and no virulence factor genes and drug resistance genes were detected in the phage. It has no toxic side effects, high safety, and is environmentally friendly, indicating that the lytic phage vB_VcaP_R25D can be used as a fungicide for aquatic Vibrio algae-lytic diseases.

[0025] The Vibrio campbellii phage vB_VcaP_R25D of the present invention was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 20, 2024, with a deposit number of GDMCC No: 65167-B1, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Postal Code: 510070. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The morphological characteristics of phages R24D and R25D. (A) Plaques of phages R24D and R25D; (B) Electron microscopic morphology of phages R24D and R25D.

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

[0028] Figure 3 The full genome map of phage R24D and R25D. The dotted boxes are differentially expressed genes.

[0029] Figure 4 Phylogenetic analysis and whole genome alignment of bacteriophages. (A) Whole genome phylogenetic tree of R24D and R25D; (B) VIRIDIC heat map of R24D and R25D; (C) Whole genome alignment of R24D, R25D, and VEN.

[0030] Figure 5 The bactericidal effect of phages. (A) Lysis curve of R24D; (B) Lysis curve of R25D; (C) Lysis curve of 1:1 mixture of R24D and R25D; (D) Lysis efficiency of different systems after co-culture for 6 hours. Each data point is the mean ± standard error of 6 biological parallels.

[0031] Figure 6The environmental stability of the phages. (A) The survival rate of phages R24D and R25D at 4°C-75°C; (B) The survival rate of phages R24D and R25D at pH 2-13. Each data point is the mean ± standard error of 3 biological replicates. DETAILED DESCRIPTION

[0032] The following examples are provided to further illustrate the present invention, but are not intended to limit the present invention. The methods and techniques used are conventional methods and techniques unless otherwise specified.

[0033] Example 1: Isolation and purification of bacteriophage

[0034] In this study, Vibrio campbellii LMG 11216 T The phage was isolated from the host Vibrio, which was purchased from the Belgian Microbial Collection Center (BCCM) as a model strain. T The culture medium was 2216E (10 M tryptone, 2 M yeast extract, artificial seawater at pH 7.5) and shaken (160 rpm min -1 , 28°C). In July 2023, water samples were collected from the wastewater pipes of Xiamen Bayishi Seafood Market and Xiashang Seafood Market for phage isolation. The water samples were filtered through a 0.22 μm polycarbonate membrane (Polycarbonate, Millipore, USA) and stored in a 4°C refrigerator in the laboratory. Before the isolation experiment, the filtered water samples were added to V. campbellii LMG 11216 in the early logarithmic growth stage. T Incubate in a shaking incubator (160 rpm min -1 , 28℃) for 24h and then filtered and sterilized to achieve the effect of enriching potential phages. Use SM buffer (5.844g NaCl, 2.4647g MgSO4·7H2O, 6.057g Tris, 0.1g Gelatin, pH=7.5, 1L distilled water, 110℃ high pressure steam sterilization for 30min) to gradient dilute the filtrate, then separate the phages by double-layer plate method, and observe the formation of plaques. Pick a single plaque for at least 5 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 1mL SM buffer for subsequent use to obtain the purified phage stock solution, which is refrigerated at 4℃ away from light.

[0035] Example 2: Enrichment of phage

[0036] Inoculate 1 mL of purified phage into 50 mL of host bacterial solution in the early logarithmic growth stage. Co-culture until the culture solution becomes clear or produces floccules and other lysis phenomena. Remove cell debris by centrifugation (10,000 × g, 4 ° C, 15 min). Filter the supernatant through a 0.22 μm filter membrane and inoculate it into 200 mL of host bacterial solution in the early logarithmic growth stage. Repeat the above steps to recover the filtrate and gradually expand the culture system to 1 L. Add 1 μg·mL of phage to 1 L of host-phage co-culture solution. -1 DNase I and RNase A were added to digest for 1 hour at room temperature to degrade free DNA and RNA in the phage suspension. Then NaCl was added to a final concentration of 1 mol / L, and after fully dissolving, it was placed at 4°C (or ice bath) for about 1 hour to promote the phage on the fragments to fall off and enter the suspension. The supernatant was collected by centrifugation (10,000×g, 4°C, 15min) and filtered through a 0.22μm filter membrane to obtain 1L of phage filtrate. 10% (wt / vol) polyethylene glycol 8000 (PEG 8000) was added to the filtrate, and it was allowed to stand at 4°C in the dark for 1-3 days. After obvious flocculent sedimentation, it was centrifuged (12,000×g, 4°C, 15min), and the supernatant was discarded and 6mL SM buffer was added to resuspend the precipitate. Cesium chloride (CsCl) density gradient centrifugation was performed, and 2 mL of CsCl solution with densities of 1.7, 1.5, and 1.3 g / mL was slowly added along the wall of the centrifuge tube. Finally, the phage concentrate was added to the upper layer, and the solution was balanced with SM buffer and ultracentrifuged (200,000×g, 4°C, 24 h). The blue or bluish-white concentrated phage band in the middle was aspirated, and the CsCl was removed by centrifugation (5,000×g, 4°C, 5 min) through a 30 kDa ultrafiltration tube (Millipore, USA). After dialyzation, SM buffer was added and stored at 4°C in the dark.

[0037] Example 3: Morphological observation of bacteriophage

[0038] Phage morphology was observed under electron microscope using phosphotungstic acid negative staining method. 10 PFU·mL -1 The phage concentrate was dropped onto a 200-mesh copper grid covered with a carbon film. After adsorption in the dark for 10-30 minutes, the sample 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 Electro Optics Laboratory Co., Tokyo, Japan) at a voltage of 120 kV, and the image was collected using a GATAN INC CCD image transmission system.

[0039] In this study, V. campbellii LMG 11216 was used T Two phages, vB_VcaP_R24D and vB_VcaP_R25D, were isolated from seafood aquaculture wastewater samples collected from Xiashang Seafood Market (118.10°E, 24.55°N) and Bashi Seafood Market (118.07°E, 24.46°N) as hosts. They are referred to as R24D and R25D. The two phages were observed to have rapid plaque formation using the double-layer plate method. Clear and transparent circular plaques with a diameter of about 1 mm can be observed with the naked eye after about 5 hours of culture, and the diameter of the plaques gradually increases with the extension of the culture time ( Figure 1 A) The expansion rate of R24D plaque diameter within 12 hours was about 2.1 μm·h -1 The final diameter is 4-5 mm, while the R25D plaque is slightly smaller, with an expansion rate of about 0.67 μm·h -1 The final diameter is 3mm. The plaques formed by the two phages are clear and transparent, and the Vibrio in the plaque area is almost completely lysed, reflecting that the two phages have extremely strong lytic properties. In addition, there is a semi-transparent halo around the plaques of both phages ( Figure 1 A), which is often considered to be related to the polysaccaride depolymerase in the phage. TEM morphology results showed that both phages R24D and R25D are short-tailed phages ( Figure 1 B), consisting 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-contractile tail (with rich tail filament structure).

[0040] Example 4: Chloroform sensitivity assay of bacteriophage

[0041] Chloroform sensitivity assays can be used to assess the presence of lipid-associated compounds in phage capsids. 8 PFU·mL -1 The phage solution was mixed with chloroform to give final concentrations of 0%, 2%, and 20%, respectively. The mixture was shaken for 30 seconds, kept at room temperature in the dark for 30 minutes, and then centrifuged at low speed (5,000 × g, 4°C, 5 minutes) to separate the phases. 2 μL of the supernatant was dropped on V. campbellii LMG 11216 T The plate was placed on a plate, allowed to stand for cooling, and then placed in a 30°C constant temperature incubator for culture. After plaques were formed, the plaques were observed. Three replicates were set for each treatment, and SM buffer was used instead of phage solution as a negative control.

[0042] The results of the chloroform sensitivity test showed that there was no significant difference in the titrated plaques of the different chloroform concentration treatment groups, and the plaque morphology, size, plaque appearance speed and turbidity were all consistent, indicating that the two phage strains R24D and R25D were insensitive to chloroform, did not contain lipid substances in the capsid, and had no lipid membrane wrapped around the capsid.

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

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

[0045] The one-step growth curve can be used to explore the incubation period and lysis amount of phages, that is, the time required for phages to adsorb, invade, assemble and release progeny phages, as well as the average number of progeny phages released by each infected host cell. The one-step growth curve results showed that the incubation period of both phages was less than 10 minutes, indicating that the two phages quickly completed replication and assembly in the host cells and quickly lysed their host cells ( Figure 2 During the phage release process, R24D (32±2PFU·cell -1 ) was always greater than R25D (17±1 PFU·cell -1 )(P<0.01), and its lysis amount was nearly twice that of R25D. In summary, both phages followed a life strategy of short incubation period and small burst, and the lysis amount of R24D was higher than that of R25D.

[0046] Example 6: Host lysis spectrum of bacteriophage

[0047] In order to determine the host lysis spectrum of bacteriophages R24D and R25D and compare the differences in their host ranges, a total of 26 strains of Vibrio were selected for the experiment, including 4 strains of Vibrio kannerii, 4 strains of Vibrio harveyi, 2 strains of Vibrio erwensii, 4 strains of Vibrio alginolyticus, 9 strains of Vibrio parahaemolyticus, 1 strain of Vibrio anguillarum, 1 strain of Vibrio fluvialis and 1 strain of Vibrio brasiliensis isolated from the hepatopancreas of farmed shrimp. In this study, the plaque titration method was used to detect 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, poured onto a 2216E solid culture plate and cooled and solidified. Take 2 μL of phage liquid (infectivity of about 10 8 PFU·mL -1 ) was dropped onto the Vibrio plate after cooling and solidification, and then placed in a 30°C constant temperature incubator for culture to observe the formation of plaques. Each treatment was repeated 3 times, and the host Vibrio campbellii LMG 11216 was used. T Instead of the tested strain, it was used as a positive control, and SM buffer was used as a negative control.

[0048] The Vibrio species used in the host range test in this study include Vibrio campbellii, Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Vibrio erwinii, Vibrio brasiliensis and Vibrio fluvii, which are common in aquaculture environments. Their isolation environments cover a variety of habitats such as aquaculture water bodies and aquatic animal tissues. The host range results showed that both phages R24D and R25D showed a narrow host lysis range, and among the tested Vibrio species, they almost only infected the isolated host V. campbelliiLMG 11216 T , indicating that the two phages showed strong host specificity (Table 1). However, this study cannot rule out the possibility that the two phages infect other untested hosts.

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

[0050]

[0051]

[0052] Note: “+” indicates that it can be infected, and “-” indicates that it cannot be infected.

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

[0054] The phage pure strain DNA was extracted by phenol-chloroform extraction. 1.5 μL of 100 mg mL -1 Proteinase K solution, 100 μL of 10% (wt / vol) SDS solution, 10 μL of 0.5 mol·L-1 EDTA solution, mix well and place in a 55℃ constant temperature water bath for digestion for 3h. Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1, volume ratio) mixture to the digested phage suspension, mix well and centrifuge (12,000×g, 4℃, 5min), transfer the upper aqueous phase to a new centrifuge tube, and repeat this step twice. An equal volume of chloroform-isoamyl alcohol mixture (24:1, volume ratio) was added to the collected upper aqueous phase solution, and the mixture was fully shaken for 30 seconds. After centrifugation (12,000×g, 4°C, 5 min), the upper aqueous phase solution was collected and mixed with an equal volume of isopropanol liquid. After mixing, the mixture was allowed to stand at -20°C for at least 1 hour and then centrifuged again (12,000×g, 4°C, 5 min). The phage nucleic acid precipitate was collected, rinsed twice with pre-cooled 70% ethanol solution and air-dried. The nucleic acid precipitate was then dissolved in 100 μL TE buffer (10 mmol·L -1 Tris-HCl, 1mmol·L -1 EDTA, pH = 8.0) and stored at -20°C. The phage genome sequencing was completed by Shanghai Hanyu Biotechnology Co., Ltd. (China). The sequencing library was completed by Ultra TM The DNA library was constructed using a DNA library preparation kit (NEB, USA) and sequenced using the Illumina HiSeq 4000 platform.

[0055] Example 8: Phage genome analysis

[0056] The raw data and assembled genomes 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 website on the Galaxy server (http: / / galaxy.pasteur.fr). The phage genome was predicted by GeneMarkS (http: / / exon.gatech.edu / GeneMark / index.html) online software and ORF Finder online software (https: / / www.ncbi.nlm.nih.gov / orffinder / ). The BLASTP search algorithm of the non-redundant (NR) protein database of the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov) was used with an E-value ≤ 10. -5Each translated ORF was searched and compared with the threshold, and the ORFs were annotated and functionally predicted according to the protein functions encoded by the 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 / ). The virulence genes of phages were detected in the Virulence Factor Database of Pathogens (VFDB, http: / / www.mgc.ac.cn / VFs / main.html). Antibiotic resistance genes were detected in the Comprehensive Drug Resistance Database (CARD, https: / / card.mcmaster.ca / analyze / rgi). The lifestyle of phages was predicted using the online platform Phage AI based on the whole phage genome.

[0057] Both phages R24D and R25D have linear double-stranded DNA with a total length of 44,615bp and 43,710bp, respectively, and G+C contents of 43.69% and 43.74%, respectively. PhageTerm was used to identify the phage ends and predict the packaging machinery. Both phages R24D and R25D belong to COS (Cohesive End Sites) packaging machinery. R24D has a 357bp direct terminal repeat sequence, and R25D has a 334bp direct terminal repeat sequence. The ORFs of phages R24D and R25D were predicted by GenemarkS. R24D has 51 ORFs. Except for the first ORF on the negative strand, the rest of the ORFs are all on the positive strand. The predicted 51 ORFs were compared by BLAST to predict gene functions (Table 2), of which 24 (47.05%) functional genes were annotated; R25D has 48 ORFs, all of which are on the positive strand, of which 22 (45.83%) functional genes were annotated. The genes of the two phages were classified according to the annotated functions, which can be roughly summarized as structural protein, DNA metabolism, DNA assembly, host lysis and auxiliary metabolism related genes, and the rest are hypothetical protein related genes ( Figure 3 ). In addition, no tRNA, virulence factors, and drug resistance-related genes were detected in either phage strain.

[0058] Among the known functional genes of the two phages, about 26% are related to DNA metabolism, including RNA polymerase (T7-like RNA polymerase), DNA helicase (RecA-like domain of Escherichia colibacteriophage T7 Gp4Dhelicase), DNA polymerase I (Polymerase I), DNA ligase (DNAligase), exonuclease (5'-3'exonuclease), and endonuclease (Endonuclease VII) related genes; about 50% of the genes are related to structure and assembly, such as terminase large subunit and terminase small subunit, as well as abundant structural proteins, such as head-tail connecting protein, head scaffolding protein, capsid and scaffold protein, tail tubular protein, tail protein, internal virion protein, tail fiber protein, and endonuclease VII. 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. At the same time, both phage genomes contain a 699bp gene encoding a protein containing a lysozyme domain (R24D ORF37, R25D ORF 35). There are also some auxiliary metabolic genes related to host metabolism in the genomes of the two phages, such as genes encoding the Acetyltransferase (GNAT) family and Acetyl-CoA acetyltransferase related to acetyltransferase (R24D: ORF 29, ORF 43, ORF 44, R25D: ORF 27, ORF 40, ORF 41).At the same time, phage R25D has a unique functional gene, which is predicted to be a PrgI family protein (ORF 4), and R24D has three unique functional genes (ORF4, OFRF25, ORF40), which are annotated as SAM-dependent methyltransferase, phosphomevalonate kinase, and perforin, respectively.

[0059] Table 2 ORF predicted gene functions of phage R24D and R25D

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[0064] Example 9: Phylogenetic Analysis and Classification of Bacteriophages

[0065] Phages most similar to the phage genome were identified using BLASTn (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and their sequences were downloaded from Genbank (https: / / www.ncbi.nlm.nih.gov), and genome alignment was performed using clinker (Gilchrist, 2021#105). Phage relationships were analyzed based on the online website VICTOR (https: / / ggdc.dsmz.de / victor.php) for virus classification and evolutionary tree construction, and VIRIDIC (Virus Intergenomic Distance Caculator) was used to compare the similarities between phage genomes.

[0066] Based on NCBI BLASTN analysis, a total of 11 phages showed high sequence identity with R24D and R25D (Table 3), among which the Vibrio alginolyticus phage VEN isolated from Greece (NCBI sequence number NC_047903.1) showed the highest similarity with R24D and R25D, with sequence identities of 92.32% and 92.17%, and coverage of 85% and 88%, respectively (Table 3). Based on VICTOR phylogenetic analysis, it was further confirmed that R24D and R25D were clustered with VEN, and the three phages had a close relationship ( Figure 4A). All three phages were isolated from environments related to aquatic product farming and sales. The difference is that the hosts of phages R24D and R25D were Vibrio cannelli, while the host of phage VEN was Vibrio alginolyticus ( Figure 4 A). The inter-genomic similarity analysis using VIRDIC showed that the genome similarities between R24D and R25D and VEN were 80.4% and 82.9%, respectively, and the similarity between the R24D and R25D genomes was 90.7%, indicating that R24D, R25D, and VEN belong to the same genus Trungvirus in the family Autographiviridae, but belong to different species in the genus Trungvirus ( Figure 4 B), is a new virus of the genus Trungvirus. vB_VcaP_R25D was named Vibrio campbellii phage vB_VcaP_R25D, and was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on September 20, 2024, with the deposit number GDMCCNo:65167-B1, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Postal Code: 510070.

[0067] Comparative analysis of the whole genomes of phages R24D, R25D, and VEN showed that the genomes of the three phages can be divided into genes related to structural proteins, DNA metabolism, DNA assembly, host lysis, and auxiliary metabolism according to their functions. In addition, the genes related to DNA metabolism and tail structural proteins in the three phages have high homology (91.58%-99.45%) ( Figure 4 C). By comparing the whole genomes of three closely related phages, R24D, R25D, and VEN, it was found that RNA polymeras, DNA primase, DNA polymerase, 5'-3' exonuclease, and Recombination endonuclease VII were highly homologous, indicating that the three phages had the same DNA metabolic process and pathway. In terms of tail structure, the Head-tail connecting protein and Tail tubular protein of the three phages were also highly homologous. In addition, all three phages had genes related to Cell wall hydrolase and Lysozyme domain-containing protein, indicating that all three phages were virulent phages. However, genes encoding Holin were only detected in the genomes of phages R24D and VEN, and the genome of R25D just lacked this gene encoding Holin 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 bacteriocidal efficiency of bacteriophage

[0072] To investigate the effects of two phages on the host Vibrio campbellii LMG 11216 T The lytic activity of phages was tested under different MOI conditions for bacteriocidal effects on the host. The host Vibrio campbelliiLMG 11216 was inoculated at a volume ratio of 1:50. T In 2216E liquid medium, shaker culture (160 rpm min -1 , 30℃) to the early logarithmic growth stage (OD 600 =0.1-0.2), then add bacterial solution and phages of different abundances at MOIs of 0.01, 0.1, 1, 10 and 100 to obtain a Vibrio-phage co-incubation system, and then use a multifunctional microplate reader (Synergy H1, Bio-Tek, USA) to monitor the absorbance of the samples at 600 nm in real time in each experimental well system. The co-culture was carried out for 24 hours, and the detection was performed every 30 minutes. The plate was shaken for 5 seconds before detection, and the detection temperature was 28°C. At least 5 replicates were set for each treatment, and 2216E liquid culture medium and bacterial solution were used as negative and positive controls, respectively.

[0073] Effects of bacteriophage R24D and R25D on host Vibrio campbellii LMG 11216 at different MOIs T The results of the bactericidal experiment are as follows Figure 5 By adding phage R24D, R25D or their mixture to the host Vibrio culture medium in the exponential growth phase, phage R24D, R25D and the mixture can quickly lyse the host, and the larger the MOI, the stronger the phage lysis effect on the host. In the early stage of culture, the OD of the host Vibrio can be effectively reduced to 600 Controlled within 0.4 ( Figure 5 A, 5B, 5C). After 6 hours of co-culture, the lysis effect of phage R24D, R25D and phage mixture on the growth of host Vibrio reached the strongest level. At this time, the bacteriocidal efficiency of phage under all MOI conditions (MOI of 0.001-10) reached about 90% ( Figure 5D). Further analysis showed that 6 hours after adding the phage, the bactericidal efficiency of phage R24D at MOI of 0.001-0.1 was slightly higher than that at MOI of 0.1-10, indicating that adding a small amount of phage R24D can quickly achieve a higher bactericidal effect. When the two phages were mixed and added to the host in the exponential growth phase, the lysis effect was not significantly different from that of adding phage alone, indicating that the mixing of two similar phages did not improve their bactericidal efficiency.

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

[0075] By testing the thermal stability and pH sensitivity of phages, the ability of phages to resist environmental stress can be explored. First, dilute the phage concentrate with SM buffer to obtain 10 8 PFU·mL -1 The phage solution was set aside, and the temperature gradient was set to 4℃, 15℃, 25℃, 35℃, 45℃, 55℃, 65℃ and 75℃, respectively. The phage solution was placed in different temperature incubators in the dark for 6 hours, and the samples were returned to room temperature and the phage infectivity was determined by the double-layer plate method. SM buffers with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 were prepared, and the phage solution was placed in SM buffers with different pH values. After 6 hours of incubation at room temperature in the dark, the phage infectivity was determined by the double-layer plate method. Three replicates were set for each treatment.

[0076] The environmental stability results of phage R24D and R25D are shown in Figure 6 As shown. Phage R24D and R25D can maintain high activity (survival rate> 90%) within 4℃-35℃, and their activity is significantly reduced when the temperature reaches 55℃ ( Figure 6 A). When the temperature reaches 65°C, phage R24D is completely inactivated, while phage R25D still has a certain activity under the high temperature condition of 65°C (survival rate>4.5%), and is not completely inactivated until 75°C. In comprehensive comparison, both phages R24D and R25D show good stability in the temperature range of 4°C-45°C, but the activity of phage R24D is more susceptible to high temperature and is more sensitive to temperature changes. At the same time, the optimal pH range of the two phages is 7-8, and their survival rates are as high as 95.56%-111.1%. Both phages maintain high activity in the pH range of 4-10 (survival rate>80%). Only when the pH is lower than 3 or higher than 11, the phages completely lose their activity. Data analysis showed that there was no significant difference in the survival rate of the two phage strains within the pH range of 3-10. When the pH reached 11, the survival rate of phage R25D was significantly higher than that of R24D (P<0.05), indicating that phage R25D had a stronger tolerance to alkaline environments ( Figure 6 B).

[0077] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 regarded as the protection scope of the present invention.

Claims

1. Lytic bacteriophage vB_VcaP_R25D, deposit number: GDMCC No: 65167-B1.

2. A phage composition comprising the lytic phage vB_VcaP_R25D according to claim 1.

3. Use of the lytic bacteriophage vB_VcaP_R25D 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_R25D 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_R25D 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_R25D 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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