Phage compound preparation aiming at vibrio harveyi and application

Abstract

The invention relates to the technical field of microorganisms, in particular to a bacteriophage compound preparation for vibrio harveyi and application. The compound preparation comprises a vibrio harveyi bacteriophage RDP-VB25018 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No. 46671 and a vibrio harveyi bacteriophage RDP-VB25018R with the preservation number of CGMCC No. 46672. The compound preparation can effectively overcome the problem that bacteria easily generate resistance in bacteriophage treatment, has a high-efficiency synergistic lysis effect on original strains and resistant strains of Vibrio harveyi, has a lysis spectrum width of 84%, is far superior to that of a single bacteriophage, and has a good application prospect. And one or more of vibrio orientalis, vibrio harveyi, vibrio cholerae, vibrio canbainii, vibrio Ewana, vibrio alginolyticus and vibrio parahaemolyticus can be cracked. Animal experiments show that the compound preparation can obviously reduce the death rate of infected prawns from 85% or above to about 11%, and pathogenic bacteria in tissues are thoroughly removed. The invention provides an efficient and safe solution for green prevention and control of aquatic vibriosis.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a bacteriophage complex preparation targeting Vibrio harveyi and its application. Background Technology

[0002] Vibrio, a typical representative of Gram-negative bacteria, is one of the most common bacterial groups in the marine environment, widely distributed in bays, nearshore areas, and estuaries. The genus Vibrio is diverse, with major pathogenic Vibrio species including Vibrio harveyi, Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae. With the continuous expansion of mariculture scale and the sustained increase in stocking density, outbreaks of aquaculture diseases are becoming more frequent, and the scale of disease occurrence in the aquaculture industry is showing a year-on-year upward trend.

[0003] Vibrio harveyi is a common pathogen in aquaculture, belonging to the genus Vibrio of the class Gamma-Proteobacteria. It can infect across species and is most prevalent in spring and summer, with high detection rates in fish and shrimp products such as large yellow croaker, tongue sole, mud clams, and shrimp. Animals infected with Vibrio harveyi often exhibit symptoms such as skin ulceration and bleeding, enteritis with ascites, and scabies. It can also cause acute hepatopancreatic necrosis and white spot disease in invertebrates. Furthermore, Vibrio harveyi is a zoonotic opportunistic pathogen; infection in the marine environment can cause diarrhea, abscesses, septicemia, and necrotic wound infections in humans, triggering severe inflammatory responses.

[0004] Currently, the prevention and control of vibriosis mainly relies on strategies such as antibiotic treatment, vaccination, and probiotic application. However, antibiotic overuse easily leads to the emergence of multidrug-resistant strains, vaccine protection rates are limited by serotype diversity, and the effectiveness of prevention and control varies regionally, severely limiting the application of traditional strategies. Therefore, developing non-antibiotic prevention and control strategies based on biological control and ecological regulation is particularly necessary.

[0005] Phage therapy is an effective measure to reduce or replace antibiotic resistance. However, screening for natural phages is complex and time-consuming, making it difficult to efficiently isolate phage strains capable of lysing bacteria. Simultaneously, the rapid evolution of phage resistance mechanisms can enhance the host bacteria's resistance to phages. Bacteria develop resistance through mutations (such as altering cell surface receptors or acquiring CRISPR-Cas immune systems), thereby weakening the effectiveness of phages as antibacterial drugs. If only a few types of phages are relied upon, resistant bacteria can quickly gain dominance, leading to treatment or prevention failure. Isolating new phages capable of infecting these resistant bacteria is a direct means of restoring and maintaining preventative and therapeutic effects.

[0006] To rapidly eliminate pathogens and delay the development of phage resistance, phage cocktail therapy, which combines multiple phages, can be used to improve the therapeutic effect of phage therapy. Compared to single phage therapy, phage cocktail therapy can more effectively combat infections caused by multidrug-resistant bacteria and significantly reduce the probability of pathogens developing phage resistance. However, conventional cocktails often simply mix phages from different sources, and the synergy, broad-spectrum activity, and ability to eliminate resistant strains in these combinations are largely random. There is a lack of proactive and efficient design strategies to construct phage combinations that can anticipate and overcome bacterial resistance issues.

[0007] Therefore, developing a phage composition that can actively respond to the development of phage resistance, has a clear synergistic effect, and a broader lysis spectrum is of great significance for promoting the practical application of phage therapy in aquaculture.

[0008] The information disclosed in this background section is only intended to enhance the understanding of the background technology of this application and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a phage complex formulation and its application that exhibits synergistic antibacterial activity against Vibrio harveyi and can effectively lyse the original Vibrio harveyi and its phage-resistant mutant strains.

[0010] The technical solution of this invention is as follows:

[0011] On one hand, a phage complex formulation targeting Vibrio harveyi bacteriophage, comprising Vibrio harveyi bacteriophage RDP-VB25018 and Vibrio harveyi bacteriophage RDP-VB25018R, wherein Vibrio harveyi bacteriophage RDP-VB25018 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 25, 2025, with accession number CGMCC No. 46671. Vibrio harveyi bacteriophage RDP-VB25018R was also deposited at the same center on August 25, 2025, with accession number CGMCC No. 46672.

[0012] Furthermore, the titers of both Vibrio harveyi phage RDP-VB25018 and Vibrio harveyi phage RDP-VB25018R are not less than 10. 10 PFU / mL.

[0013] Furthermore, the outbreak period of Vibrio harveyi phage RDP-VB25018 is approximately 120 min, with an outbreak quantity of 340 PFU / cell; the outbreak period of Vibrio harveyi phage RDP-VB25018R is 110 min, with an outbreak quantity of 460 PFU / cell.

[0014] Furthermore, the Vibrio harveyi phage RDP-VB25018 maintains a titer of 10 at 20℃~50℃. 8 ~10 10 PFU / mL. The Vibrio harveyi bacteriophage RDP-VB25018R maintained a titer of 10 at 20℃~50℃. 8 ~10 10 PFU / mI.

[0015] Furthermore, the titers of Vibrio harveyi phage RDP-VB25018 and Vibrio harveyi phage RDP-VB25018R are maintained at 10 at pH 5–10. 6 PFU / mL or higher.

[0016] Furthermore, the compound preparation can lyse one or more of the following strains: Vibrio orientalis, Vibrio harveyi, Vibrio cholerae, Vibrio campei, Vibrio erwinis, Vibrio alginolyticus, and Vibrio parahaemolyticus.

[0017] Furthermore, the pyrolysis rate of the composite formulation is not less than 84%.

[0018] On the other hand, the present invention provides the use of a phage complex formulation against Vibrio harveyi in the preparation of a medicament for the prevention or treatment of Vibrio harveyi infection.

[0019] On the other hand, the present invention provides the application of a phage complex formulation targeting Vibrio harveyi in the preparation of a formulation for the prevention and control of vibrio diseases in aquaculture.

[0020] Furthermore, according to the above applications, the formulation is a water disinfectant or an aquaculture environment improver.

[0021] The beneficial effects achieved by this invention are as follows:

[0022] 1. The compound formulation of this invention can effectively lyse the mutant strain VB25018R, which is resistant to bacteriophage RDP-VB25018, solving the problem that single bacteriophages are easily rendered ineffective due to bacterial resistance. In animal infection models, the compound formulation showed significant therapeutic effects against infections caused by resistant bacteria, with a mortality rate of only 12.2%.

[0023] 2. The compound formulation of this invention exhibits superior inhibitory effects against the original bacterium VB25018 compared to any single bacteriophage. Animal experiments show that the compound formulation demonstrates the best therapeutic effect against the original bacterium infection, with a mortality rate as low as 11.1%, which is superior to the single-strain treatment group.

[0024] 3. The compound formulation of this invention achieved a lysis rate of 84% against 100 test Vibrio strains, which is significantly higher than that of the two single bacteriophage strains (60% and 74%). Furthermore, it can effectively lyse various common pathogenic Vibrio species found in aquatic organisms, such as Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio cholerae.

[0025] 4. In the infection model, the compound preparation of this invention reduced the mortality rate of shrimp from over 85% to 11-12%, and the bacterial load in the tissues after treatment was close to that of healthy shrimp. Attached Figure Description

[0026] Figure 1 Colony morphology of Vibrio harveyi VB25018 on TCBS plates.

[0027] Figure 2 Plaque morphology of Vibrio harveyi phage RDP-VB25018.

[0028] Figure 3 Colony morphology of Vibrio harveyi resistance mutant strain VB25018R on TCBS plates.

[0029] Figure 4 Plaque morphology of Vibrio harveyi phage RDP-VB25018R.

[0030] Figure 5 Transmission electron micrographs of bacteriophages RDP-VB25018(A) and RDP-VB25018R(B).

[0031] Figure 6 One-step growth curves of bacteriophages RDP-VB25018 and RDP-VB25018R.

[0032] Figure 7 Effect of temperature on the activity of bacteriophage RDP-VB25018.

[0033] Figure 8 Effect of temperature on the activity of bacteriophage RDP-VB25018R.

[0034] Figure 9 Effect of pH on the activity of bacteriophages RDP-VB25018 and RDP-VB25018R.

[0035] Figure 10 In vitro growth inhibition curves of single and combined bacteriophages against Vibrio harveyi VB25018.

[0036] Figure 11 In vitro growth inhibition curves of single and combined bacteriophages against Vibrio harveyi resistant strain VB25018R. Detailed Implementation

[0037] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] In this invention, unless otherwise specified, the equipment and raw materials used are commercially available or commonly used in the field. The methods in the following embodiments, unless otherwise specified, are conventional methods in the field. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In this invention, *Vibrio harveyi* VB25018 is referred to as "host VB25018" or simply "VB25018", *Vibrio harveyi* bacteriophage RDP-VB25018 is referred to as "phage RDP-VB25018" or simply "RDP-VB25018", and *Vibrio harveyi* bacteriophage RDP-VB25018R is referred to as "phage RDP-VB25018R" or simply "RDP-VB25018R".

[0039] Example 1: Screening and identification of Vibrio harveyi bacteriophages

[0040] 1.1 Isolation and Identification of Vibrio harveyi VB25018

[0041] Shrimp samples exhibiting typical symptoms such as decreased activity, reduced feeding, and fluorescence in some dying shrimp were collected from aquaculture farms. Tissue homogenates were placed in LB liquid medium (3% NaCl) and incubated at 37°C with shaking for 12 hours. The homogenates were then streaked onto TCBS plates and incubated overnight at 37°C in a bag. Colony morphology was observed, and yellow colonies were selected for further streaking and purification twice on TCBS plates to obtain uniformly morphologically homogeneous colonies. Figure 1 As shown, dominant single colonies were selected and inoculated into 3 mL of LB liquid medium, and cultured with shaking at 160 rpm and 37°C for 4 h.

[0042] DNA extraction from the prepared bacterial suspension was performed using a bacterial DNA extraction kit from Tiangen Biotech Co., Ltd. Subsequently, PCR amplification was performed using universal 16S rDNA primers with the bacterial DNA as a template. After identification by 16S rRNA gene sequencing, it was confirmed as *Vibrio harveyi*, named VB25018, and preservation measures were taken. The *Vibrio harveyi* bacterial suspension was densely streaked onto LB agar plates and incubated upside down at 37°C for 12 hours. Then, the bacterial growth was scraped and placed in 2216E liquid medium, mixed thoroughly, and then combined with 60% glycerol broth at a 1:1 ratio and stored at -80°C.

[0043] 1.2 Isolation of Vibrio harveyi bacteriophage RDP-VB25018

[0044] (1) Wastewater treatment: The shrimp farming water and sludge were mixed and filtered, centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered through a membrane (0.22 μmol). The filtrate was mixed with the host bacterial culture in the logarithmic growth phase at a ratio of 1:1 and cultured in a shaker at 37℃ for 16 h. The supernatant was then collected and filtered through a membrane (0.22 μmol). The filtrate ① was stored at 4℃ for later use.

[0045] (2) Phage enrichment: Take 0.1 mL of VB25018 bacterial suspension and 1 mL of filtrate ① and add it to 5 mL of LB liquid medium (3% NaCl). Incubate at 160 rpm and 37℃ for 12 h on a shaker. Then centrifuge at 12,000 rpm for 10 min. Filter the supernatant through a 0.22 μm filter. This is filtrate ②. Repeat the above operation to enrich the phage a second time. Store filtrate ③ at 4℃ for later use.

[0046] (3) Phage isolation: Phage isolation was performed using the double-plate method. 0.1 mL of logarithmically growing VB25018 bacterial suspension was mixed with 0.6% LB soft agar and spread on an LB solid plate. After the soft agar solidified, 40 μL of filtrate ③ was spotted onto the plate and allowed to stand until the mixture was absorbed. The plate was then incubated in a 37℃ incubator with a bag for 6 h. A transparent phage plaque was picked up with an inoculation loop and placed in 1 mL of SM buffer. The plate was shaken for 15 s to allow the phage to be completely released, thus obtaining Vibrio harveyi phage in its preliminary form.

[0047] (4) Phage Purification: Phage purification was performed using the double-plate method. A 1:1 mixture of phage extract and host bacterial suspension was added to LB soft agar cooled to approximately 50°C, then spread onto LB solid plates and incubated at 37°C for 6 hours. A single clear plaque with smooth edges was picked and placed in 1 mL of SM buffer. This process was repeated three times until uniformly sized, smooth-edged phage plaques were obtained, thus completing phage purification. Figure 2 As shown.

[0048] (5) Preparation of bacteriophage: Take 0.1 mL of VB25018 bacterial suspension and 1 mL of bacteriophage proliferation solution and add them to 35 mL of LB liquid medium. Shake and incubate at 160 rpm and 37 ℃ for 6 h. Then centrifuge at 12,000 rpm for 10 min. Filter the supernatant through a membrane (0.22 μmol) and store it at 4 ℃ for later use.

[0049] (6) Preservation of bacteriophages: One strain of Vibrio harveyi bacteriophage was isolated and named RDP-VB25018. The bacteriophage suspension was mixed with 60% glycerol at a ratio of 1:1 and stored at -80℃.

[0050] 1.3 Isolation of Vibrio harveyi VB25018R, which is resistant to phage

[0051] (1) VB25018 bacterial culture in the logarithmic growth phase (concentration 10) 8 CFU / mL) was inoculated at a multiplicity of infection (MOI) of 0.1 to a concentration of 10 8 The culture system contained PFU / mL RDP-VB25018 bacteriophage. After the phage lysis caused the turbid bacterial suspension to become clear, surviving strains were screened using the streak plating method. Single colonies were collected for spot testing, and colonies that could still grow in the presence of RDP-VB25018 were considered resistant.

[0052] (2) Using the method described in 1.1 above, single colonies were purified, and the colony morphology on TCBS plates was as follows: Figure 3 As shown. After verification by 16S rRNA gene sequencing, the phage resistance mutant was preserved as a strain and named VB25018R. The strain was deposited and stored at -80℃.

[0053] 1.4 Isolation of anti-Vibrio phage RDP-VB25018R

[0054] Using the phage-resistant bacteriophage VB25018R as the host, a new phage, named RDP-VB25018R, was isolated according to the phage isolation method described in section 1.2 above. The plaque morphology of Vibrio harveyi phage RDP-VB25018R is as follows: Figure 4 As shown, the strain of bacteriophage was preserved.

[0055] 1.5 Electron microscopic observation of Vibrio harveyi bacteriophage

[0056] 1.5.1 Experimental Methods

[0057] Take 20 μL of liquid containing coarse phage particles and drop it onto a copper grid. Allow it to settle naturally for 15 minutes, then blot away excess liquid with filter paper from the side. Add one drop of 2% phosphotungstic acid (PTA) to the copper grid to stain the phage for 10 minutes. Then blot away the staining solution with filter paper from the side. After the sample dries, observe the phage morphology using an electron microscope. Figure 5 As shown, where Figure 5 5A is an electron micrograph of bacteriophage RDP-VB25018. 5B is an electron micrograph of bacteriophage RDP-VB25018R.

[0058] 1.5.2 Experimental Results and Analysis

[0059] Electron microscopy findings: Bacteriophage RDP-VB25018 has a polyhedral, three-dimensionally symmetrical head that encapsulates nucleic acid, with a diameter of 67 nm and a tail length of approximately 106 nm.

[0060] The bacteriophage RDP-VB25018R has a polyhedral head that encapsulates nucleic acid, with a diameter of 71 nm and a tail length of approximately 109 nm.

[0061] 1.6 Whole genome sequence sequencing of Vibrio harveyi bacteriophage

[0062] 1.6.1 Experimental Methods

[0063] Using Illumina TruSeq TM Library construction using the Nano DNA Sample Prep Kit method; the specific steps are as follows:

[0064] 1) Construct a library starting with 1 μg of bacteriophage genomic DNA;

[0065] 2) Covaris M220 ultrasonically breaks down DNA to 300-500 bp;

[0066] 3) Fill in the 3' end with A, and connect the index connector (TruSeq) TM Nano DNA Sample Prep Kit);

[0067] 4) Library enrichment, PCR amplification for 8 cycles;

[0068] 5) 2% agarose gel recovery target band (Certified Low Range Ultra Agarose);

[0069] 6) TBS380 (Picogreen) quantitative analysis: mix according to the data ratio before loading.

[0070] 7) Bridged PCR amplification was performed on the cBot solid-phase support to generate clusters;

[0071] 8) Illumina Hiseq sequencing platform, performing 2×150bp sequencing.

[0072] 1.6.2 Experimental Results and Analysis

[0073] The complete genome of bacteriophage RDP-VB25018, as shown in SEQ ID NO.1, is 77505 bp in size. The genome contains no homologous sequences of drug resistance genes or virulence factors, indicating high biosafety.

[0074] The complete genome of bacteriophage RDP-VB25018R, as shown in SEQ ID NO.2, is 71417 bp in size. The genome contains no homologous sequences of drug resistance genes or virulence factors, and has high biosafety.

[0075] Example 2: Fermentation Performance Analysis

[0076] 2.1 Determination of Vibrio harveyi phage titer

[0077] 2.1.1 Experimental Methods

[0078] Take 0.1 mL of the logarithmic growth phase bacterial suspension and phage concentrate, respectively, and add them to 10 mL of LB soft agar (dissolved and cooled to room temperature). Mix well and pour onto LB solid plates (completely covering a 9 cm plate), then incubate at 37°C for 6 hours. Next, collect a single spot and place it in 1 mL of SM buffer, shake thoroughly, and filter through a 0.22 μm filter. Take 0.1 mL of the filtrate and serially dilute it 10-fold with sterile water. Then, take 0.1 mL of the bacterial suspension and the diluted phage solution, respectively, and add them to 10 mL of LB soft agar. Mix well and spread onto LB solid plates, then incubate at 37°C for 6 hours.

[0079] Select a countable plate, count the phage plaques, and calculate the phage titer (PFU / mL) as the number of plaques × dilution factor / sample volume (mL).

[0080] 2.1.2 Experimental Results and Analysis

[0081] The titer of bacteriophage RDP-VB25018 (host bacterium RDP-VB25018) was determined to be 3.2 × 10⁻⁶. 10 The titer of bacteriophage RDP-VB25018R (host bacterium RDP-VB25018R) was 7.6 × 10⁻⁶ PFU / mL. 10 PFU / mL.

[0082] 2.2 Determination of the optimal multiple of infection (MOI) of Vibrio harveyi bacteriophage

[0083] 2.2.1 Experimental Methods

[0084] The concentration of the host bacteria cultured to the stable phase was adjusted to 10. 8 CFU / mL. MOIs were set at 0.001, 0.01, 0.1, 1, 10, and 100. Phage solutions with determined titers were added to the host bacterial culture in the specified proportions, mixed thoroughly, and incubated at 160 rpm and 28°C with shaking for 6 h. The culture was then centrifuged at 12,000 rpm for 5 min, and the supernatant was used to determine the phage titer using the double-plate method. The highest possible multiplicity of infection (MOI) is the optimal MOI.

[0085] 2.2.2 Experimental Results and Analysis

[0086] Table 1. Phage titers (PFU / mL) under different multiplicity of infection conditions.

[0087]

[0088] The results are shown in Table 1. When the multiplicity of infection (MCI) was 0.1, the titer of phage RDP-VB25018 was the highest, at 3.2 × 10⁻⁶. 10 PFU / mL; the highest titer of phage RDP-VB25018R was 7.6 × 10⁻⁶ when the multiplicity of infection was 0.01. 10 PFU / mL. Based on these results, it is inferred that RDP-VB25018R has a stronger adsorption capacity for the host and can adsorb and infect the host more efficiently.

[0089] 2.3 One-step growth curve of Vibrio harveyi bacteriophage

[0090] 2.3.1 Experimental Methods

[0091] Take phage fluid and host bacterial fluid separately (10) 6 Mix 0.5 mL of phage (CFU / mL) at the ratio required for the optimal multiple of infection (MOI = 0.1), incubate at room temperature for 10 min, then centrifuge at 12,000 rpm for 10 min, discard the supernatant, wash with LB liquid medium, and centrifuge again at 12,000 rpm for 1 min. Repeat the above operation, washing the precipitate 3 times, then add 10 mL of LB liquid medium preheated to 37°C, mix thoroughly, and quickly incubate in a shaker at 37°C. Take a sample of 0.2 mL every 10 min initially and thereafter, centrifuge at 12,000 rpm for 2 min, and use 0.1 mL of the supernatant to determine the phage titer. Finally, plot a one-step growth curve with infection time on the x-axis and the logarithm of the phage titer in the infection system on the y-axis.

[0092] 2.3.2 Experimental Results and Analysis

[0093] like Figure 6As shown, bacteriophage RDP-VB25018 infected the host bacteria within 30 minutes, and the number of bacteriophages did not increase significantly, indicating that the phage incubation period was 30 minutes. From 30 to 150 minutes after infecting the host bacteria, the number of bacteriophages increased rapidly. This period is the phage outbreak period, which is about 120 minutes, and the phage outbreak amount is about 340 PFU / cell. In the following 30 minutes, the number of bacteriophages remained unchanged, that is, the phage reached the stationary growth period.

[0094] The incubation period of phage RDP-VB25018R was 20 min, the outbreak period was 110 min, the outbreak quantity of phage was approximately 460 PFU / cell, and the number of phages did not change significantly in the following 30 min.

[0095] The results above show that both phage strains exhibited large burst volumes and good lysis, making them suitable for phage therapy. In comparison, phage RDP-VB25018R had a shorter latency period and a similar burst duration, but produced a larger burst volume.

[0096] 2.4 Determination of the optimal growth temperature for bacteriophages

[0097] 2.4.1 Experimental Methods

[0098] Take a sterile 2mL centrifuge tube, add 1mL of phage stock solution, and incubate at 20℃, 28℃, 37℃, 50℃, 60℃ and 70℃ for 30min, 60min and 90min respectively. After the incubation time is over, take out the sample tube, dilute to the appropriate concentration, and take 0.1mL of the treated phage solution and the host bacterial solution respectively. Use the double plate method to determine the phage titer.

[0099] 2.4.2 Experimental Results and Analysis

[0100] like Figure 7 As shown, phage RDP-VB25018 exhibited stable activity within the temperature range of 20℃ to 50℃, maintaining a titer of 10⁸–10¹⁰ PFU / mL. When the temperature was further increased to 60℃, partial inactivation of the phage began to occur with prolonged exposure time. At 70℃, phage activity decreased sharply within 30 minutes and was completely lost within 60 minutes.

[0101] The thermal stability of bacteriophage RDP-VB25018R showed a trend generally consistent with that of RDP-VB25018, such as... Figure 8 As shown, bacteriophage RDP-VB25018R maintains high titer below 50℃. However, when the temperature reaches 60℃ to 70℃, partial and complete inactivation gradually occur.

[0102] This indicates that the above-mentioned bacteriophages have good stability at 20–50°C and grow best at 28–37°C.

[0103] 2.5 pH stability determination of Vibrio harveyi bacteriophage

[0104] 2.5.1 Experimental Methods

[0105] Add 3 mL of LB liquid culture medium to each 5 mL sterile test tube, adjusting the pH to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 respectively. Then place the tubes in a 37°C constant temperature water bath. After temperature equilibration, add 0.1 mL of phage stock solution and host bacterial solution respectively, and incubate at 37°C for 1 hour. Determine the phage titer using the double-layer plate method. Before measurement, adjust the pH to approximately 7.0 using hydrochloric acid or sodium hydroxide solution.

[0106] 2.5.2 Experimental Results and Analysis

[0107] like Figure 9 As shown, phages RDP-VB25018 and RDP-VB25018R maintained high activity and titers at 10 within a pH range of 5–10. 6 PFU / mL or higher. Under strongly acidic or alkaline conditions with a pH below 4 or above 10, the phage activity drops sharply until it is completely inactivated. The optimal pH for both of the above-mentioned phage strains is 7.0, and they exhibit some tolerance to acidic and alkaline environments.

[0108] Example 3: Verification of the antibacterial effect of Vibrio harveyi bacteriophage

[0109] 3.1 Experimental Methods

[0110] Bacterial suspension in the logarithmic growth phase was collected, and phage suspension and bacteria were co-incubated in 96-well plates (MOI = 0.1). A control sample without any phage suspension was prepared simultaneously. The 96-well plates were placed in a microplate reader and incubated at 37°C in shaking mode. OD600 values ​​were automatically measured hourly for 24 hours. OD600 values ​​were recorded, growth curves were plotted, and inhibition rates were calculated.

[0111] Antibacterial rate (24h) = (OD600 of control group - OD600 of experimental group) / OD600 of control group × 100%

[0112] This was used to quantify the inactivation ability of a single bacteriophage (RDP-VB25018 or RDP-VB25018R) or a mixture of bacteriophages (RDP-VB25018 and RDP-VB25018R) against Vibrio harveyi VB25018 and VB25018R.

[0113] 3.2 Experimental Results and Analysis

[0114] Depend on Figure 10 It was observed that VB25018 grew normally, with its OD600 increasing from approximately 0.1 to 1.2 within 24 hours, exhibiting a typical S-shaped growth curve. RDP-VB25018 inhibited the growth of VB25018 6 hours after inoculation, with OD600 consistently below 0.6, indicating that RDP-VB25018 had a good inhibitory effect on VB25018-sensitive strains. However, under the influence of RDP-VB25018R, OD600 growth was slow, with a final OD600 of approximately 0.8 after 24 hours, indicating that RDP-VB25018R partially inhibited VB25018, but the effect was not as strong as RDP-VB25018. When a mixture of two phages was inoculated, the OD600 remained below 0.4 after inoculation and did not recover within 24 hours, demonstrating a superior inhibitory effect compared to a single phage.

[0115] Depend on Figure 11 It was observed that VB25018R and VB25018 exhibited similar growth patterns, both displaying S-shaped growth curves. When RDP-VB25018 was used for infection, the OD600 growth curve was similar to that of the control group, indicating that RDP-VB25018 had no inhibitory effect on the resistant strain VB25018R. However, when a mixture of RDP-VB25018R and bacteriophage was used for infection, OD600 began to inhibit the growth of VB25018R 4 hours after inoculation and remained at a low level thereafter, demonstrating a significant inhibitory effect. This indicates that bacteriophages containing RDP-VB25018R can effectively inhibit resistant strains.

[0116] The above results indicate that phage RDP-VB25018 effectively inhibits the susceptible strain VB25018, while phage RDP-VB25018R specifically inhibits strains resistant to RDP-VB25018. The mixture of the two phages exhibits a synergistic inhibitory effect on both susceptible and resistant strains, effectively preventing the development of resistance. Furthermore, the antibacterial effect of the phage mixture is superior to that of a single phage.

[0117] Example 4: Lysis spectrum of Vibrio harveyi bacteriophage

[0118] 4.1 Experimental Methods

[0119] The lytic effects of Vibrio harveyi phage RDP-VB25018, phage RDP-VB25018R, and phage mixtures on Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio cholerae, and other Vibrio species were determined using the double-layer plate method. One hundred Vibrio strains were randomly selected from the strain library, and the bacterial suspensions were revived. Then, 0.1 mL of the bacterial suspension and phage suspension were taken separately, mixed, and added to LB soft agar, mixed again, and quickly poured into LB solid medium plates. After cooling and solidification, the plates were incubated upside down at 37°C for 6 hours. The appearance of phage plaques was observed to determine the success of lysis.

[0120] 4.2 Experimental Results and Analysis

[0121] Table 1. Lysis effect of Vibrio harveyi bacteriophage

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128] Where: "+" represents pyrolysis

[0129] Table 1 shows that bacteriophage RDP-VB25018 could infect 60 Vibrio strains with a lysis rate of 60%. Bacteriophage RDP-VB25018R could infect 74 Vibrio strains with a lysis rate of 74%. The bacteriophage mixture could infect 84 Vibrio strains with a lysis rate of 84%. Furthermore, the bacteriophages used showed varying degrees of lysis against multiple pathogenic Vibrio species. These results indicate that mixing the two bacteriophages maintained their lysis stability while expanding their host range and enhancing their lysis effect.

[0130] Example 5: Treatment experiment of Vibrio harveyi bacteriophage on Litopenaeus vannamei

[0131] Healthy whiteleg shrimp with an average weight of 5.0 ± 0.5 grams were selected and temporarily raised for one week to acclimatize. They were then infected with Vibrio harveyi VB25018 and VB25018R respectively, and the activated strains were prepared to a concentration of 10... 6The bacterial suspension was prepared at CFU / mL. The experiment consisted of 7 groups, as shown in Table 2, with 3 replicates per group and 30 shrimp per replicate. Except for the control group, shrimp in all other groups were immersed in the corresponding bacterial suspension for 2 hours. After infection, the shrimp were transferred to the corresponding phage suspension or sterile seawater for continuous immersion treatment. Normal feed was provided during the experiment.

[0132] Table 2 Animal Experiment Grouping Information

[0133] Group Infection Management Treatment CON Not infected Immersed in sterile seawater S Immersion infection with VB25018 strain Immersed in sterile seawater R Immersion infection with VB25018R strain Immersed in sterile seawater PS Immersion infection with VB25018 strain Two hours after infection, immerse in RDP-VB25018 suspension. PR Immersion infection with VB25018R strain Two hours after infection, immerse in RDP-VB25018R suspension. PMix-S Immersion infection with VB25018 strain Two hours after infection, the sample was immersed in a phage mixture suspension. PMix-R Immersion infection with VB25018R strain Two hours after infection, the sample was immersed in a phage mixture suspension.

[0134] Wherein: CON: blank control group; S: infection control group; R: infection control group; PS: RDP-VB25018 treatment group; PR: RDP-VB25018R treatment group; PMix-S: phage mixture treatment group; PMix-R: phage mixture treatment group.

[0135] The shrimp were continuously observed and recorded for 96 hours, with the number of dead shrimp recorded every 24 hours, and the cumulative mortality rate (%) was calculated. At the end of the experiment (96 hours), 5 shrimp were randomly selected from each group of surviving shrimp, and their hepatopancreatic tissue was homogenized, spread on TCBS agar plates, and the number of Vibrio harveyi colonies (CFU / g tissue) was counted.

[0136] Table 3. Cumulative mortality rate (%, Mean±SD) over 96 hours

[0137]

[0138] According to the results in Table 3

[0139] (1) Validation of the virus attack model

[0140] In the infection control groups (S group and R group): 96 hours after infection, the cumulative mortality rates were as high as 86.7% and 90.0%, respectively, showing a highly significant difference compared to the blank control group (CON group, 3.3%). This result demonstrates that both the original bacterium VB25018 and its resistant mutant strain VB25018R successfully established lethal infection models, proving the reliability of the experimental system.

[0141] (2) Single phage therapy has significant effects

[0142] Single-plant treatment groups (PS group and PR group):

[0143] In the PS group (using RDP-VB25018) against the original bacterial infection, the 96-hour mortality rate decreased to 18.9%, and the protection rate was 78.2% (vs. S group).

[0144] In the PR group (using RDP-VB25018R) against resistant bacterial infections, the 96-hour mortality rate decreased to 20.0%, and the protection rate was 77.8% (vs. R group).

[0145] Conclusion: Both bacteriophage strains showed clear and effective therapeutic effects on their respective host bacteria (original bacteria or resistant bacteria), significantly reducing the mortality rate from over 85% to around 20%.

[0146] (3) Phage complex formulations exhibit remarkable synergistic advantages

[0147] Combined therapy groups (PMix-S group and PMix-R group):

[0148] In the PMix-S group targeting the original bacterial infection, the 96-hour mortality rate further decreased to 11.1%, and the protection rate increased to 87.2%.

[0149] In the PMix-R group against resistant bacterial infections, the 96-hour mortality rate further decreased to 12.2%, and the protection rate increased to 86.4%.

[0150] Key comparisons:

[0151] For primary bacterial infections, the combination therapy (PMix-S, 11.1%) was significantly more effective than the corresponding single phage therapy (PS, 18.9%).

[0152] For infections caused by resistant bacteria, the combination therapy (PMix-R, 12.2%) was also significantly more effective than the corresponding single phage therapy (PR, 20.0%).

[0153] Conclusion: The combined treatment was superior to the best single-strain treatment group in treating both strains of infection, with the lowest mortality rate.

[0154] (4) Dynamic process analysis: Early manifestation of synergistic effect

[0155] Observe the 24-hour mortality rate:

[0156] More than 20% of the infected control group (S, R) experienced acute death.

[0157] The mortality rate in all treatment groups (PS, PR, PMix-S, PMix-R) was kept at a very low level (3.3%-6.7%), indicating that the phages acted rapidly.

[0158] Observe for 48-96 hours:

[0159] The mortality rate growth curve in the combined treatment groups (PMix-S, PMix-R) was consistently the flattest, ultimately showing a significant difference of approximately 7-8 percentage points compared to the monoclonal treatment groups. This indicates that the synergistic effect is not only reflected in the final outcome but also in the ability to sustain infection control and prevent late-stage mortality.

[0160] In summary, the mortality rates of both the S and R control groups after infection were over 85%, indicating the success of the challenge model. Monotherapy with RDP-VB25018 and RDP-VB25018R (PS and PR groups) showed significant therapeutic effects, reducing the mortality rate to approximately 20%. The phage mixture treatment groups (PMix-S and PMix-R) demonstrated even better therapeutic effects against both strains than the monotherapy groups, further reducing the mortality rate to approximately 10%, exhibiting the best protective effect.

[0161] Vibrio load calculations in hepatopancreatic tissues showed a significant reduction in bacterial counts in all treatment groups compared to the infected control group. Similarly, compared to the single-strain phage treatment group, the phage mixture groups (PMix-S and PMix-R) demonstrated more thorough bacterial clearance, with tissue loads approaching those of the blank control group and significantly lower than the single-strain treatment group. This confirms the ability of phages to effectively proliferate and lyse pathogens in vivo.

[0162] Table 4 Results of organ and tissue bacterial load experiments (lg(CFU / g))

[0163]

[0164] As shown in Table 4

[0165] (1) The infection model was successfully established.

[0166] In the infection control groups (S group and R group): the Vibrio harveyi load in the hepatopancreatic tissue was extremely high, reaching 7.52 and 7.48 lg (CFU / g) respectively.

[0167] Compared with the blank control group (CON group, 3.01lg(CFU / g)), the bacterial load in the infection group increased by nearly 4.5 orders of magnitude (approximately 10,000 times), which conclusively proves that the pathogens achieved massive colonization and proliferation in the main target organs after challenge, and the infection model was successful.

[0168] (2) Single phage therapy can effectively eliminate pathogens.

[0169] Single-plant treatment groups (PS group and PR group):

[0170] The bacterial load in the PS group (treated with RDP-VB25018 for the original bacterial infection) was 4.111g (CFU / g).

[0171] The bacterial load in the PR group (treated with RDP-VB25018R for resistant bacterial infection) was 4.251g (CFU / g).

[0172] Evaluation of clearance efficacy: The bacterial load in both monophage treatment groups was significantly reduced by more than 3.2 orders of magnitude (approximately 99.9%) compared to the corresponding infection control group (~7.51g (CFU / g)). This demonstrates that single phage treatment can effectively clear the corresponding pathogens in the hepatopancreas.

[0173] (3) The phage complex formulation exhibited optimal pathogen clearance ability and approached the level of healthy cells.

[0174] Combined therapy groups (PMix-S group and PMix-R group):

[0175] The bacterial load in the PMix-S group (combination therapy for primary bacterial infection) was 3.271g (CFU / g).

[0176] The bacterial load in the PMix-R group (combination therapy for resistant bacterial infections) was 3.531g (CFU / g).

[0177] Key comparisons and conclusions:

[0178] Superior to monoclonal treatment: The bacterial load in the compound preparation group (3.27, 3.53) was significantly lower than that in the corresponding monoclonal treatment group (4.11, 4.25), indicating that the compound preparation was more effective in clearing pathogens in organs.

[0179] Recovery to near-healthy state: The bacterial load in the compound preparation group was extremely close to that of the blank control group (CON, 3.01). In the PMix-S group, the bacterial load was only 0.26 log units higher than that in the healthy group (i.e., less than 2 times), which in actual treatment can be regarded as the pathogens being basically eliminated and the tissue load being restored to near the physiological baseline.

[0180] (4) Data correlation: corroboration with mortality results

[0181] This data is highly consistent with the mortality results (Table 3), forming a complete chain of evidence:

[0182] Infected control group: High bacterial load (~7.51g CFU / g) corresponds to high mortality (>85%).

[0183] Single-strain treatment group: moderate bacterial load (~4.21 g CFU / g) corresponds to moderate mortality (~20%).

[0184] The combined preparation group: low bacterial load (~3.41g CFU / g) corresponds to low mortality (~11%).

[0185] In summary, this animal experiment demonstrates that bacteriophages RDP-VB25018 and RDP-VB25018R can effectively treat Vibrio harveyi infection in Litopenaeus vannamei, significantly reducing shrimp mortality and the number of pathogens in the body. RDP-VB25018 and RDP-VB25018R exhibit specific therapeutic effects against certain strains (sensitive or resistant), and the combined use of the two bacteriophages produced a significant synergistic effect, demonstrating superior therapeutic efficacy against both strains compared to a single bacteriophage, exhibiting a broader antibacterial spectrum and stronger protective efficacy.

[0186] The embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A phage complex formulation targeting Vibrio harveyi, characterized in that: The compound formulation comprises Vibrio harveyi phage RDP-VB25018 and Vibrio harveyi phage RDP-VB25018R. Vibrio harveyi phage RDP-VB25018 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 25, 2025, with accession number CGMCC No. 46671; Vibrio harveyi phage RDP-VB25018R was deposited at the same center on August 25, 2025, with accession number CGMCC No. 46672.

2. The phage complex formulation against Vibrio harveyi according to claim 1, characterized in that: The titers of both Vibrio harveyi phage RDP-VB25018 and Vibrio harveyi phage RDP-VB25018R are not less than 10. 10 PFU / mL.

3. The phage complex formulation against Vibrio harveyi according to claim 1, characterized in that: The outbreak period of Vibrio harveyi phage RDP-VB25018 was approximately 120 min, with an outbreak rate of 340 PFU / cell; the outbreak period of Vibrio harveyi phage RDP-VB25018R was 110 min, with an outbreak rate of 460 PFU / cell.

4. The phage complex formulation against Vibrio harveyi according to claim 1, characterized in that: The titers of Vibrio harveyi phage RDP-VB25018 and Vibrio harveyi phage RDP-VB25018R were maintained at 10 at 20℃~50℃. 8 ~10 10 PFU / mL.

5. The phage complex formulation against Vibrio harveyi according to claim 1, characterized in that: The Vibrio harveyi phage RDP-VB25018 and Vibrio harveyi phage RDP-VB25018R maintained a titer of 10 at pH 5–10. 6 PFU / mL or higher.

6. The phage complex formulation against Vibrio harveyi according to claim 1, characterized in that: The compound preparation can lyse one or more of the following strains: Vibrio orientalis, Vibrio harveyi, Vibrio cholerae, Vibrio campbellii, Vibrio erwinis, Vibrio alginolyticus, and Vibrio parahaemolyticus.

7. The phage complex formulation against Vibrio harveyi according to claim 1, characterized in that: The pyrolysis rate of the compound formulation is not less than 84%.

8. The use of the phage complex formulation against Vibrio harveyi according to claim 1 in the preparation of a medicament for the prevention or treatment of Vibrio harveyi infection.

9. The application of the phage complex preparation against Vibrio harveyi according to claim 1 in the preparation of a preparation for the prevention and control of vibrio diseases in aquaculture.

10. The application according to claim 8, characterized in that: The preparation is a water disinfectant or an aquaculture environment improver.

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