Lytic bacteriophage vB_vpaS_r40z and its application in prevention and control of vibrio parahaemolyticus in aquaculture environment

Abstract

The application discloses a lytic bacteriophage vB_VpaS_R40Z and application thereof in preventing and controlling Vibrio parahaemolyticus in aquaculture environment. The Vibrio parahaemolyticus bacteriophage vB_VpaS_R40Z has a preservation number of GDMCC No: 67440-B1. The R40Z can lyse multiple strains of Vibrio parahaemolyticus and Vibrio alginolyticus, and has the ability to adapt to the aquaculture environment, and remains high activity within the range of 4-25 DEG C and pH 4-10; genome analysis does not detect virulence genes or drug resistance genes; phylogenetic comparison with the whole genome shows that the similarity of R40Z with the related bacteriophage is lower than the species level threshold, and R40Z is a new bacteriophage species; R40Z can significantly inhibit the growth of the host under multiple infection multiplicities. R40Z has the advantages of strong lytic ability, good environmental stability, high genome safety and unique evolutionary characteristics, and is a water product vibrio disease biological prevention and control bacteriophage with application potential.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the lytic bacteriophage vB_VpaS_R40Z and its application in the prevention and control of Vibrio parahaemolyticus in aquaculture environments. Background Technology

[0002] Vibriosis is caused by Vibrio spp. ( Vibrio Infectious diseases in aquatic animals caused by bacteria can manifest as explosive mortality or persistent low-level infection, thus being considered one of the most common and harmful bacterial diseases in global aquaculture. Vibrio parahaemolyticus (V. parahaemolyticus) Vibrio parahaemolyticus Vibrio parahaemolyticus (VPC) is a halophilic, facultative anaerobic, Gram-negative bacterium widely found in brackish or semi-brackish water environments such as marine, estuarine, and coastal sediments. It is not only a major pathogen causing foodborne gastroenteritis in humans but is also considered a primary pathogenic factor in various aquatic animal diseases, such as shrimp and fish. During aquaculture, VPC can invade the host through the body surface, gills, or digestive tract, inducing septicemia, enteritis, and hepatopancreatic necrosis, leading to large-scale mortality. In shrimp farming, VPC is responsible for Acute Hepatopancreatic Necrosis Disease (AHPND) and High Lethality Vibrio disease. Vibrio Vibrio vulnificus disease (AVVD) is one of the major pathogens. Existing research indicates that AHPND can cause a cumulative mortality rate of up to 100% in Litopenaeus vannamei within approximately 10–35 days after infection; while HLVD is more common in the late larval stages of shrimp, with mortality rates exceeding 90% within 24–48 hours of infection. Related studies indicate that AHPND outbreaks have significantly impacted several major shrimp-producing countries in Asia and Latin America, placing continuous pressure on the stability and economic benefits of the industry. The high incidence and rapid spread of vibrio vulnificus disease highlight the vulnerability of current aquaculture systems in disease control, necessitating the development of safer, more effective, and sustainable alternative control methods.

[0003] Currently, the main measure for controlling vibriosis in aquaculture is still the use of antibiotics. However, long-term improper use of antibiotics can lead to drug resistance in Vibrio, resulting in decreased drug efficacy, which in turn requires increasing the dosage and frequency of antibiotic use, creating a vicious cycle that accelerates the spread of drug-resistant strains. This not only increases the difficulty of disease control but also poses environmental and food safety risks. Against this backdrop, bacteriophages, as viruses capable of specifically infecting and lysing bacteria, have attracted widespread attention. Phage therapy relies on its highly specific recognition and infectivity of pathogenic bacteria, achieving efficient and precise bactericidal effects by invading and lysing the host bacteria. Compared with traditional antibiotic therapy, phage therapy has several advantages: its high specificity allows it to target specific pathogenic strains without disrupting the host's normal flora, thus reducing side effects such as flora imbalance; at the same time, phages can interact with bacteria during evolution, maintaining their ability to inhibit drug-resistant strains through natural selection and adaptive mutations, while antibiotics often gradually become ineffective due to the accumulation of resistance. In addition, phages are widely distributed in nature, and their application usually does not cause environmental accumulation and pollution, possessing high ecological safety. Based on these characteristics, bacteriophages are considered to have unique advantages in dealing with bacterial infections, especially drug-resistant infections, and have broad application prospects. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a lytic bacteriophage vB_VpaS_R40Z and its application in the prevention and control of Vibrio parahaemolyticus in aquaculture environments.

[0005] This invention is achieved through the following technical solutions:

[0006] The first objective of this invention is to provide a Vibrio parahaemolyticus phage strain ( Vibrio parahaemolyticus Phage vB_VpaS_R40Z was deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67440-B1.

[0007] This invention uses Vibrio parahaemolyticus (Vibrio parahaemolyticus) V. parahaemolyticus Using vp-201911 as the host strain, the lytic bacteriophage vB_VpaS_R40Z (R40Z) was isolated from wastewater in the sewage system of the Shekou Seafood Market in Shenzhen. Through systematic analysis of its morphological characteristics, infection dynamics, host range, environmental adaptability, and bactericidal ability, combined with whole-genome annotation and phylogenetic comparison, the safety, genetic characteristics, and application potential of R40Z in disease control were evaluated.

[0008] A second object of the present invention is to provide a phage composition comprising the above-mentioned Vibrio parahaemolyticus phage vB_VpaS_R40Z.

[0009] A third object of the present invention is to provide the use of the above-mentioned Vibrio parahaemolyticus phage vB_VpaS_R40Z or phage composition in killing and / or preventing Vibrio alginolyticus, Vibrio parahaemolyticus or Vibrio kanehirae.

[0010] A fourth object of the present invention is to provide the use of the above-mentioned Vibrio parahaemolyticus phage vB_VpaS_R40Z or phage composition in the preparation of a medicament against Vibrio alginolyticus, Vibrio parahaemolyticus or Vibrio kanehirae.

[0011] A fifth object of the present invention is to provide the use of the above-mentioned Vibrio parahaemolyticus phage vB_VpaS_R40Z or phage composition in the preparation of disinfectants, aquatic feeds or feed additives that are resistant to Vibrio alginolyticus, Vibrio parahaemolyticus or Vibrio kanehirae.

[0012] Preferably, the Vibrio kanehirae is Vibrio kanehirae (Vibrio kanehirae). V. campbellii HJ-2.

[0013] A sixth object of the present invention is to provide a drug for treating Vibrio alginolyticus, Vibrio parahaemolyticus, or Vibrio kanehirae, comprising the aforementioned Vibrio parahaemolyticus bacteriophage vB_VpaS_R40Z or a bacteriophage composition.

[0014] A seventh object of the present invention is to provide a disinfectant, aquatic feed or feed additive that is resistant to Vibrio alginolyticus, Vibrio parahaemolyticus or Vibrio kanseri, containing the aforementioned Vibrio parahaemolyticus bacteriophage vB_VpaS_R40Z or a bacteriophage composition.

[0015] The eighth object of the present invention is to provide a method for in vitro prevention and treatment of Vibrio alginolyticus, Vibrio parahaemolyticus or Vibrio kanehirae infection, which is to apply the above-mentioned Vibrio parahaemolyticus bacteriophage vB_VpaS_R40Z or a bacteriophage composition.

[0016] Preferably, the Vibrio kanehirae is Vibrio kanehirae (Vibrio kanehirae). V. campbellii HJ-2.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention is based on V. parahaemolyticusThe host strain was vp-201911, and the lytic bacteriophage vB_VpaS_R40Z (R40Z) was isolated from wastewater in the Shekou Seafood Market in Shenzhen. R40Z forms well-defined plaques, and transmission electron microscopy (TEM) shows it has a typical long-tailed structure. Its latency period is approximately 40 min, and its burst yield reaches 138 ± 11 PFU / cell. Host range analysis indicates that R40Z can lyse multiple strains of Vibrio parahaemolyticus and Vibrio alginolyticus. Temperature and pH stability tests show that R40Z maintains high activity within the range of 4–25°C and pH 4–10, demonstrating its ability to adapt to aquaculture environments. The R40Z genome is 58,318 bp in length and contains 88 ORFs. Genome analysis did not detect any virulence or resistance genes. Phylogenetic and genome-wide comparisons showed that R40Z exhibited similarity to closely related bacteriophages below the species-level threshold and possessed specific variations in key structural proteins, indicating that it is a novel bacteriophage species. Bactericidal experiments further confirmed that R40Z significantly inhibited host growth under various infection multiples. In summary, R40Z possesses advantages such as strong lytic ability, good environmental stability, high genomic safety, and unique evolutionary characteristics, making it a promising bacteriophage for the biocontrol of vibrio parahaemolyticus diseases in aquaculture. This invention not only enriches the resources of Vibrio parahaemolyticus bacteriophages but also provides new candidate bacteriophages for the subsequent construction of aquatic pathogen bacteriophage libraries and the development of precision biocontrol systems.

[0019] Vibrio parahaemolyticus phage ( Vibrio parahaemolyticus Phage vB_VpaS_R40Z was deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67440-B1. Attached Figure Description

[0020] Figure 1 The results show the morphological characteristics and chloroform sensitivity of bacteriophage R40Z, including (A) bacteriophage plaques of bacteriophage R40Z; (B) electron micrograph of bacteriophage R40Z; and (C) chloroform sensitivity results of bacteriophage R40Z.

[0021] Figure 2 This is the host-range evolutionary tree in Example 5.

[0022] Figure 3 This is the one-step growth curve of phage R40Z in Example 6.

[0023] Figure 4 The results are the environmental stability results of phage R40Z in Example 7, including (A) pH stability results and (B) temperature stability results.

[0024] Figure 5 The results are the bactericidal effect evaluation results of phage R40Z in Example 8, including (A) the bactericidal curve of phage R40Z; and (B) the bactericidal efficiency of phage R40Z.

[0025] Figure 6 This is the genome map of phage R40Z in Example 9.

[0026] Figure 7 The results are the phylogenetic analysis and whole-genome alignment of phage R40Z in Example 10, including (A) the whole-genome phylogenetic tree of phage R40Z; (B) the VIRIDIC heatmap of phage R40Z; and (C) the whole-genome alignment of phage R40Z with similar phages. Detailed Implementation

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0028] Example 1: Isolation and purification of bacteriophages

[0029] Experimental method: Vibrio parahaemolyticus V. parahaemolyticus vp -The isolation of bacteriophages was carried out using NCBI serial numbers NZ_CP150862 and NZ_CP150863 as the host. The sample used for phage isolation was from the sewer system of the Shekou Seafood Market in Shenzhen (longitude 113°56′45.5″, latitude 22°29′23.14″). The sewer water sample (Millipore, MA, USA) was filtered through a 0.22 μm filter for phage isolation. The host bacteria were placed in nutrient-rich RO medium (10 M peptone, 2 M yeast extract, artificial seawater, pH 7.5) and cultured at 28°C and 150 rpm / min until the exponential growth phase. 1 mL of the filtered water sample was added to 20 mL of the exponentially growing Vibrio culture, and the mixture was incubated in a shaker at 28°C for 24 h. The resulting co-culture solution was filtered through a 0.22 μm filter and stored at 4°C. Phages were isolated and purified using the double-layer plate method. 3 μL of filtrate was added to different regions of the double-layer plate, and 3 μL of SM Buffer (5.844 g NaCl, 2.4647 g MgSO4·7H2O, 6.057 g Tris-HCl, 0.1 g Gelatin, 1 L distilled water, pH=7.5) was added to the blank as a control. The plates were incubated at 28℃ until plaques appeared. Individual plaques were collected and stored in 1 mL of SM buffer (the aforementioned SM Buffer). After suspending the phage in a refrigerator at 4°C for 24 h, the phage was purified using the double-layer plate method. The specific method is as follows: Take 990 μL of SM Buffer into a centrifuge tube, add 10 μL of the suspension to the centrifuge tube and perform serial dilutions, adding 1 mL of host bacterial culture to each tube, incubate in the dark for 15 min, then add 5 mL of nutrient-rich RO medium containing 0.5% (w / v) agar, and incubate at 28°C until phage plaques appear. Collect a single phage plaque and store it in 1 mL of SM Buffer. This completes one round of purification. Repeat the above steps five times to complete the purification of the phage.

[0030] Experimental results: using V. parahaemolyticus vp - Using 201911 as the host bacterium, bacteriophage R40Z was successfully isolated from a sewage sample using the double-layer agar plate method. The plaques were tracked and photographed over time, such as... Figure 1 As shown in (A), phage plaques can be clearly observed starting at 8 h, exhibiting a serrated expansion pattern. At this time, the plaque diameter is 61.54 ± 1.88 μm. After 24 h, the diameter reaches 70.13 ± 1.65 μm, and the plaque diameter expansion rate from 8 to 24 h reaches 0.53 μm·min. -1Meanwhile, the number of particles in the plaques was minimal. Over the following 24 hours, there were no significant changes in plaque size or particle size, although plaque transparency decreased slightly. Under magnification, the outer edges of the plaques were clear, without halos.

[0031] Example 2 Chloroform Sensitivity Test of Bacteriophage R40Z

[0032] Experimental Methods: To investigate whether the outer capsid of bacteriophages contains lipid components and whether its surface is coated with a lipid membrane, purified bacteriophage R40Z suspensions of the same abundance (i.e., those purified in Example 1) were taken, and 0, 20, 200, and 400 μL of chloroform were added to them respectively. After thorough mixing, the samples were incubated in the dark for 30 minutes and then centrifuged (5000×g, 4℃, 5 min). The chloroform-treated bacteriophages were detected using the double-layer plate method to observe whether there were significant changes in the morphology and number of plaques.

[0033] Experimental results: such as Figure 1 As shown in (C), the chloroform sensitivity test results indicate that the infectivity of phage R40Z is not affected by chloroform, suggesting that the outer capsid of this phage does not contain lipids.

[0034] Example 3: Amplification and enrichment of bacteriophage R40Z

[0035] To obtain a high-abundance phage suspension, phage amplification and enrichment are necessary. First, the purified phage R40Z suspension (i.e., the one purified in Example 1) is inoculated into a host cell in the early logarithmic growth stage. V. parahaemolyticus vp - (201911), and gradually scale up the culture until a 1 L culture system is reached. DNase and RNase (final concentration 1 μg / mL) are added to the culture medium. -1To decompose free nucleic acids, NaCl was added to promote the detachment of bacteriophages adsorbed on Vibrio fragments. The culture medium was then centrifuged (8000×g, 4℃, 15 min), and PEG 8000 was added to the supernatant to a final concentration of 100 g / L. The sample was incubated overnight at 4℃ to induce phage aggregation and precipitation. After a second centrifugation (10,000×g, 4℃, 60 min), the phage precipitate was eluted with SM Buffer and collected, and impurities were washed away with chloroform. Next, the sample was processed using CsCl density gradient ultracentrifugation for 4 hours (140,000×g, 4℃) to separate and collect viral bands, thus obtaining enriched virus solution. The enriched virus solution was centrifuged using a 30 kDa ultrafiltration tube (5000×g, 4℃, 5 min) to remove excess cesium chloride solution. The virus solution was washed multiple times with SM buffer and finally dialyzed with 2-3 times its volume of SM buffer to completely remove impurities. After completing the above steps, the treated virus solution was stored in a refrigerator at 4°C.

[0036] Example 4: Observation using transmission electron microscopy (TEM)

[0037] Experimental Methods: The morphological characteristics of bacteriophage R40Z were observed using a tungsten filament transmission electron microscope (TEM). A glow discharge apparatus was used to hydrophilize the carbon support membrane to enhance the adsorption capacity of the sample on the carbon membrane. Subsequently, 3 μL of dialysis-treated bacteriophage fluid (obtained by centrifugation and dialysis in Example 3) was added dropwise to the carbon support membrane and allowed to stand for 3 min to allow for full adsorption of bacteriophage R40Z. After adsorption, excess liquid on the carbon support membrane was blotted off with filter paper. After the carbon support membrane dried, phosphotungstic acid solution was added for negative staining and immediately blotted dry with filter paper; phosphotungstic acid solution was added again, negatively stained for 1-2 minutes, and then blotted dry with filter paper and allowed to air dry completely. Finally, the sample was observed using a JEM-1230 transmission electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 80.0 kV, and images were acquired using a digital charge-coupled device (CCD) camera to obtain morphological information of bacteriophage R40Z.

[0038] Experimental results: TEM observations showed that, as Figure 1 As shown in (B), bacteriophage R40Z belongs to the long-tailed bacteriophage family. Its head has an icosahedral structure and a long, flexible, non-retractable tail fiber. Its head is approximately 82.01 ± 1.40 nm long and 62.09 ± 1.63 nm wide, while its tail is approximately 143.46 ± 5.25 nm long.

[0039] Example 5: Determination of the host range of bacteriophage R40Z

[0040] The infectivity of isolated bacteriophage R40Z against Vibrio was determined by titration.

[0041] Experimental Methods: Thirty Vibrio strains were selected as host range test subjects, covering different species and ecological environments (including various related populations such as Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio kansei, and Vibrio harveyi). Vibrio were cultured to the logarithmic growth phase. 1 mL of bacterial culture was mixed with 5 mL of nutrient-rich RO medium containing 0.5% agar (w / v), and the mixture was inverted onto the surface of the lower plate to prepare a double-layer plate. After the soft agar solidified, 3 μL of purified phage R40Z suspension (obtained in Example 1) was added to each of the defined areas, and 3 μL of SM buffer was added to the blank area as a negative control. After drying, the plates were inverted and incubated at 28 ℃ to observe plaque formation and determine the infectivity of phages to different Vibrio strains. To further evaluate the efficiency of plating (EOP) of phages on different hosts, the phage suspension was divided into 10... 3 -10 8 The PFU / mL phage suspension was serially diluted, and the diluted phage suspension was dropped onto the corresponding host bilayer plate to observe the phage infection of the host.

[0042] Experimental results: Bacteriophage R40Z was able to lyse 9 of the strains, including 6 strains of Vibrio alginolyticus. V. alginolyticus D61_T3_15_W4、 V. alginolyticus HJ-1, V. alginolyticus WPAGA6 V. alginolyticus WPAGA3 V. alginolyticus ZWAG4004 V. alginolyticus Chan03, 2 strains of Vibrio parahaemolyticus V. parahaemolyticus chan02、 V. parahaemolyticus Vp-201911 and one strain of Vibrio cannibalus V. campbellii HJ-2 ( Figure 2 (See Table 1). The above results indicate that R40Z has a certain host spectrum breadth within the Vibrio genus, and in particular, it has a stable lytic ability against multiple strains of Vibrio alginolyticus.

[0043] Table 1 Host Infection Spectrum of Bacteriophage R40Z

[0044] .

[0045] Example 6: Plotting a one-step growth curve

[0046] A one-step growth curve was constructed to understand the pyrolysis cycle and pyrolysis rate of R40Z.

[0047] Experimental Methods: The host bacteria were cultured to the exponential growth phase, and purified bacteriophage R40Z suspension (obtained in Example 1) was added to achieve a multiplicity of infection (MOI) of approximately 0.01. 20 mL of the mixture was adsorbed in the dark for 5 min, centrifuged (5000×g, 4℃, 5 min), the supernatant was discarded, and the suspension was resuspended in 20 mL of nutrient-enriched RO medium. This step was repeated twice. The precipitate was added to 100 mL of nutrient-enriched RO medium and mixed thoroughly. The suspension was then placed in a 28℃ constant-temperature shaker at 160 rpm for 1 min. -1 Incubate continuously with shaking in the dark. Take samples every 10 minutes and determine phage titer using the double-layer plate method until the experiment reaches 90 minutes. Three parallel control groups were set up. Plot a one-step growth curve of phage with incubation time as the x-axis and phage titer as the y-axis.

[0048] Experimental results: Figure 3 The figure shows the growth curve of R40Z at MOI = 0.01. As can be seen from the figure, the incubation period of R40Z virus is relatively long, up to 40 min. The lysis period is from 40 to 60 min, and it enters the plateau phase after 60 min. The burst yield is about 138±11 PFU per cell.

[0049] Example 7 Environmental stability of bacteriophage R40Z

[0050] Experimental methods:

[0051] 1. Thermal stability:

[0052] A purified phage R40Z suspension of known abundance (i.e., the one purified in Example 1) was statically incubated at different temperatures, and the change in phage titer was detected by the bilayer plate method to assess its thermal stability. 5 mL of phage suspension with consistent abundance (approximately 10 mL) was taken... 4 PFU mL -1 Phages were incubated at 4℃, 25℃, 35℃, 45℃, 55℃, and 65℃, respectively. Initial abundance was measured at 0 h, and samples were taken at 3 h, 24 h, and 48 h. Phage titers were determined and survival rates were calculated using the double plate method. Three parallel experiments were set up for each treatment temperature.

[0053] 2. pH stability:

[0054] A purified phage R40Z suspension of known abundance (i.e., the one purified in Example 1) was statically incubated at different pH values, and the pH stability was assessed by detecting changes in phage titer using the bilayer plate method. SM buffer solutions with pH values ​​ranging from 2 to 12 were prepared using a precision pH meter, HCl, and NaOH solutions. 5 mL of phage suspensions of uniform abundance (approximately 10 mL) was then used. 4 PFU mL -1 The phages were incubated in different pH buffers as described above. Initial abundance was measured at 0 h, and samples were taken at 3 h, 24 h, and 48 h. Phage titers were determined and survival rates were calculated using the double plate method. Three parallel experiments were set up for each treatment.

[0055] Experimental results: The environmental stability of bacteriophage R40Z is as follows: Figure 4 R40Z maintains high activity at 4℃~25℃, covering the daily storage and transportation temperature range, which is conducive to preservation. It loses activity after incubation at 55℃ for 3 hours and is completely inactivated when the temperature rises to 65℃. R40Z maintains high activity in the pH range of 4~10, and is more stable between 5~7, covering the pH range of aquaculture environments, and can survive for a long time.

[0056] Example 8: Determination of sterilization efficiency

[0057] Experimental method: The optical density (OD) at a wavelength of 600 nm was measured using a multifunctional microplate reader. 600 To evaluate the lytic ability of bacteriophages against host Vibrio parahaemolyticus at different multiples of infection (MOIs), MOI values ​​were set to 0.01, 0.1, 1, 10, and 100. Before the experiment, freshly prepared host Vibrio cultures were added to 96-well plates and then infected with purified bacteriophage R40Z suspension (obtained in Example 1). A control group was also included, inoculated only with host Vibrio. The 96-well plates were placed in a microplate reader and cultured with shaking at 30°C. The OD of the cultures was monitored in real time using a microplate reader. 600 Data was recorded every 30 minutes for 24 hours. Five replicates were used for each treatment to ensure data reliability.

[0058] Experimental results: such as Figure 5 As shown, phage R40Z exhibited activity against bacteria in the early stages of culture (0-4 h). V. parahaemolyticus The cleavage effect of VP-201911 is observed, and the higher the MOI, the more significant the cleavage effect: when the MOI is 100, the OD... 600 The value drops rapidly and remains at a low level; when MOI is 10, OD 600The decreasing trend of the value slowed slightly, but it was still lower than that of the control group (Vibrio parahaemolyticus culture only); when the MOI was 1, 0.1, and 0.01, the OD 600 Although the values ​​decreased, the rate of decrease decreased sequentially, with the curves for the MOI=0.01 group and the control group being relatively close in the early stage. During the 4-10 h culture period, the OD values ​​for each MOI group... 600 The values ​​continue to change. OD values ​​for MOI=100 and 10 groups. 600 The value tends to stabilize, remaining between 0.8 and 1.0, and its sterilization efficiency can reach about 80%; the OD of the group with MOI=1 600 The values ​​increased slowly, but remained lower than the control group; the OD values ​​of the MOI=0.1 and 0.01 groups were... 600 The rate of increase in OD values ​​gradually accelerated, and the difference between the group with MOI=100 gradually narrowed. After 10-24 h of culture, the OD values ​​of the MOI=100 group... 600 The values ​​fluctuated slightly but remained generally stable at around 1.0; the OD values ​​for groups with MOI=10 600 The value slowly rose to around 1.2; the OD of the MOI=1 group 600 The value continued to rise, approaching 1.4 after 24 h of culture; the OD of the group with MOI=0.1 600 The value rose to around 1.6; the OD of the group with MOI=0.01 600 The OD value increased rapidly, reaching approximately 1.8 after 24 hours of culture, compared to the control group (cultured only with Vibrio parahaemolyticus, OD value was lower). 600 The values ​​were close to 1.6-1.7. At 15 h of culture, the sterilization efficiency of each group remained at around 50%, while the low MOI group showed stronger sterilization efficiency at 24 h.

[0059] Overall, phage R40Z is effective against... V. parahaemolyticus vp - 201911 exhibits strong lytic activity. When the MOI is high, the lytic effect is significant in the early stage of culture, effectively inhibiting the growth and proliferation of host Vibrio. When the MOI is low, it still has a strong antibacterial effect in the later stage of culture, and can inhibit the growth and proliferation of host Vibrio for a long time.

[0060] Example 9: Genomic characteristics of bacteriophage R40Z

[0061] Experimental methods:

[0062] 1. Extraction of bacteriophage DNA:

[0063] DNA from bacteriophages was extracted using the phenol-chloroform extraction method. 1 mL of high-abundance phage solution that had undergone dialysis (i.e., the solution obtained by centrifugation and dialysis in Example 3) was taken, and 10 μL of proteinase K solution (final concentration 20 mg / mL) was added sequentially. -1 ), 100 μL SDS solution (final concentration 10% wt vol)-1 Add 10 μL of EDTA solution (0.5 M, pH 8.0). Vortex thoroughly and digest in a 55 °C metal bath for 3 hours. After digestion, add an equal volume of phenol-chloroform-isoamyl alcohol mixture (25:24:1) to the phage digest, mix thoroughly, and centrifuge (12,000 × g, 4 °C, 10 min). Carefully transfer the upper aqueous phase to a new centrifuge tube and repeat the above steps twice. Next, add an equal volume of chloroform-isoamyl alcohol mixture (24:1) to the collected upper aqueous phase, mix, and centrifuge (12,000 × g, 4 °C, 10 min). Transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, mix, and then incubate at -20 °C for precipitation. After precipitation, centrifuge (12,000 × g, 4 °C, 10 min). After centrifugation, discard the supernatant, gently wash the nucleic acid precipitate with 500 μL of pre-cooled 70% ethanol solution, and centrifuge (12,000 × g, 4 °C, 10 min). Repeat this step twice. Finally, discard the supernatant, and after the nucleic acid precipitate has completely dried, add 50 μL of TE buffer (10 mmol / L). -1 Tris-HCl, 1 mmol / L -1 DNA was resuspended in EDTA (pH 8.0). DNA concentration and quality can be detected by agarose gel electrophoresis or Nanodrop (Thermo Fisher Scientific, MA, United States).

[0064] 2. Phage genome analysis:

[0065] The complete genomes of the bacteriophages used were sequenced and assembled by Shanghai Hanyu Biotechnology Co., Ltd. (China). Sequencing libraries were constructed using the NEBNext® Ultra™ DNA Library Prep Kit, and paired-end high-throughput sequencing was performed on the Illumina HiSeq4000 platform. After quality control with Trimmomatic v0.32, raw data were analyzed and the genome assembled using Velvet v1.2.03. The DNA packaging mechanism and genome ends of the bacteriophages were identified using the online website PhageTerm on the Galaxy server (http: / / galaxy.pasteur.fr). Open reading frames (ORFs) were predicted using GeneMarkS (http: / / topaz.gatech.edu / GeneMark / genemarks.cgi) and ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ). ORFs were annotated and their functions predicted using the BLASTP search algorithm on the Non-Redundant (NR) protein database of the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov). tRNA genes were identified using tRNAscan-SE v2.0 (http: / / lowelab.ucsc.edu / tRNAscan-SE / ). Virulence genes of bacteriophages were detected using the Virulence Factor Database (VFDB, http: / / www.mgc.ac.cn / VFs / main.html). Antibiotic resistance genes of bacteriophages were detected using the Comprehensive Antibiotic Resistance Database (CARD, https: / / card.mcmaster.ca / analyze / rgi). The lifestyle of bacteriophages was predicted using the PhageAI online platform (http: / / www.PhageAI.com). Interactive visualizations of genomic functional modules and their distributions were generated using the Proksee tool (https: / / proksee.ca / ).

[0066] Experimental Results: The bacteriophage possesses a linear double-stranded DNA molecule with a length of 58,318 bp and a GC content of 46.19%. It contains 88 open reading frames (ORFs). Among these genes, 51 had no matching information found in the NCBI database, and most of them belonged to hypothetical proteins. Of the 37 identified ORFs, 14 genes are involved in nucleic acid synthesis, replication, and repair processes, including genes related to DNA ligase, primase, DNA polymerase, DNA polymerase beta subunit, endonuclease, and exonuclease. The structural and packaging modules include 15 genes such as capsid protein, tail protein, virion structural protein, and terminase large subunit.

[0067] Example 10: Phylogenetic and Genomic Analysis Comparison of Bacteriophage R40Z

[0068] Experimental Methods: Nucleotide sequences were aligned using BLASTn (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to identify phages similar to the R40Z genome. Sequences were downloaded from GenBank (https: / / www.ncbi.nlm.nih.gov), and Clinker was used to analyze genomic collinearity and similarity. Phylogenetic relationships of the phages were analyzed using the online website VICTOR (https: / / ggdc.dsmz.de / victor.php), which constructs viral classification and phylogenetic trees. The similarity between phage genomes was further validated using the viral genome distance calculator VIRIDIC (https: / / rhea.icbm.uni-oldenburg.de / viridic / ).

[0069] Experimental Results: Based on NCBI BLASTN nucleotide alignment results, a total of 12 bacteriophage genome sequences showed homology with vB_VpaS_R40Z. However, these bacteriophages exhibited significant differences in host range, ecological origin, and geographical distribution, with hosts including Vibrio parahaemolyticus (VPS). V. parahaemolyticus ), Vibrio harveyi ( V. harveyi ), sodium-dependent Vibrio ( V. natriegens ) and Vibrio lysinensis ( V. coralliilyticus These bacteriophages were collected from various environments, including seafood market wastewater, domestic sewage, estuary waters, surface seawater, and shellfish, covering regions such as China, South Korea, the United States, and Mexico.

[0070] Although R40Z shares sequence similarity with vB_VpaS_AL_2 and vB_VpS_BA3, its phylogenetic tree shows that R40Z clusters independently and does not belong to the same branch as any previously reported phages. Further VIRIDIC genome similarity calculations revealed that, despite some similarity between R40Z and vB_VpaS_AL_2 and vB_VpS_BA3, their genomic identity remains below the phage "species" threshold (≥95%), indicating a clear uniqueness in their overall genome structure.

[0071] Collinear alignment of the genomes of 12 related bacteriophages revealed that while R40Z maintained high overall structural similarity with some other bacteriophages, it exhibited significant differences in several key functional regions, particularly tail assembly proteins and viral particle proteins (corresponding to ORF41 and ORF42). These proteins are involved in crucial steps such as tail formation, host recognition, and genetic material injection, and variations in these proteins typically lead to functional differentiation in host specificity, infection mechanisms, or ecological adaptations. This characteristic further supports the determination of R40Z as an independent bacteriophage species.

[0072] Phage R40Z was named Vibrio parahaemolyticus phage. Vibrio parahaemolyticus Phage vB_VpaS_R40Z was deposited on December 8, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China, with accession number GDMCC No: 67440-B1.

[0073] discuss

[0074] 1) This invention uses Vibrio parahaemolyticus V. parahaemolyticus Using vp-201911 as the host strain, the lytic bacteriophage R40Z was successfully isolated from wastewater in the sewage system of the Shekou Seafood Market in Shenzhen. Its plaques formed clearly visible transparent areas within approximately 8 hours, indicating that the bacteriophage possesses strong lytic ability.

[0075] 2) One-step growth curves showed that R40Z had a latency period of approximately 40 min and a burst rate of 138 ± 11 PFU / cell, significantly higher than vB_VpaS_AL_2, which is most similar to it in genome (latency period of 10 min, burst rate of 68 PFU / cell). R40Z adopted an infection strategy with a longer latency period but a higher burst rate, which usually means that it has a stronger proliferative capacity in a liquid environment, which is conducive to achieving efficient proliferation and effective lysis after establishing infection.

[0076] 3) Before bacteriophages are used for disease control in aquatic organisms, genomic safety is a key indicator. Ideally, applied bacteriophages should not carry virulence-related genes or antibiotic resistance genes (ARGs) to avoid potential toxic effects or the risk of resistance transmission. Genomic analysis results show that R40Z does not contain virulence factors or resistance genes, has a simple genome structure, and meets the safety requirements for aquatic applications.

[0077] 4) Environmental stability tests confirmed that R40Z maintained high activity within the range of 4–25℃, and only became completely inactive after treatment at 55℃ for 3 hours. It also exhibited good stability within the pH range of 4–10, with the optimal conditions being pH 6–7. Its tolerance range is highly consistent with common aquaculture environments (24–30℃, pH 6–8), indicating that R40Z has good survival and lysis capabilities under actual aquaculture conditions.

[0078] 5) At the genomic level, the full-length linear double-stranded DNA of R40Z is 58,318 bp, with 88 predicted ORFs, a significant portion of which are hypothetical proteins. Its structural modules include typical structural genes such as capsid proteins, tail-related proteins, and the large subunit of the terminator enzyme, but it exhibits specific sequence differences in key regions related to host recognition and tail assembly (such as tail assembly proteins and viral particle proteins). These differences typically reflect the independence of bacteriophages in infection mechanisms, host adaptation, or ecological functions, providing a genomic basis for the unique biological characteristics of R40Z.

[0079] 6) Phylogenetic and whole-genome comparisons showed that although R40Z was similar to vB_VpaS_AL_2 and vB_VpS_BA3 at the sequence level, its similarity was below the species level threshold and it had specific variations in key structural proteins, indicating that it was a new bacteriophage species.

[0080] In summary, R40Z exhibits strong lytic activity, good environmental stability, clear genetic safety, and a characteristic genome structure, demonstrating its potential application value in the prevention and control of Vibrio parahaemolyticus infection in aquaculture.

Claims

1. Vibrio parahaemolyticus phage ( Vibrio parahaemolyticus phage)vB_VpaS_R40Z, accession number: GDMCC No: 67440-B1.

2. A phage composition containing the Vibrio parahaemolyticus phage vB_VpaS_R40Z as described in claim 1.

3. The use of the Vibrio parahaemolyticus phage vB_VpaS_R40Z of claim 1 or the phage composition of claim 2 in the preparation of a drug for treating Vibrio alginolyticus, Vibrio parahaemolyticus or Vibrio kanehirae.

4. The use of the Vibrio parahaemolyticus phage vB_VpaS_R40Z of claim 1 or the phage composition of claim 2 in the preparation of disinfectants, aquatic feeds or feed additives that are resistant to Vibrio alginolyticus, Vibrio parahaemolyticus or Vibrio kanehirae.

5. The application according to claim 3 or 4, characterized in that, The Vibrio cannibalus mentioned is Vibrio cannibalus HJ-2.

6. A drug for treating Vibrio alginolyticus, Vibrio parahaemolyticus, or Vibrio kanehirae, characterized in that, Contains the Vibrio parahaemolyticus phage vB_VpaS_R40Z of claim 1 or the phage composition of claim 2.

7. A disinfectant, aquatic feed, or feed additive resistant to Vibrio alginolyticus, Vibrio parahaemolyticus, or Vibrio kanehirae, characterized in that, Contains the Vibrio parahaemolyticus phage vB_VpaS_R40Z of claim 1 or the phage composition of claim 2.

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