Acid-resistant broad-spectrum vibrio parahaemolyticus phage LVPP58 and application thereof in prevention and control of vibriosis

Abstract

The application belongs to the field of aquatic animal bacterial disease prevention and control, and discloses an acid-resistant broad-spectrum Vibrio parahaemolyticus phage LVPP58 and application of the phage in Vibrio disease prevention and control, the preservation number of the phage is CCTCC M 2025322. The phage is a broad-spectrum Vibrio parahaemolyticus phage, and can not only lyse Vibrio parahaemolyticus, but also lyse Vibrio alginolyticus. The phage LVPP58 is stable in pH 3-11 and 30-60 DEG C, the phage can produce a synergistic effect when used in combination with antibiotics, can enhance the treatment effect, and can reduce the incidence of drug resistance, and provides a new strategy for coping with the problem of bacterial drug resistance.

Description

Technical Field

[0001] This invention relates to the field of bacterial disease control in aquatic animals, specifically to an acid-resistant broad-spectrum Vibrio parahaemolyticus phage LVPP58 and its application in the control of vibrio diseases. Background Technology

[0002] Vibrio parahaemolyticus, belonging to the genus Vibrio, is a halophilic Gram-negative bacterium with a single flagellum at the end of its cell. It does not produce spores or capsules. As an important opportunistic pathogen affecting both humans and animals, it can cause various aquatic animal diseases, including skin hemorrhage and tail rot in fish, white spot disease in abalone feet, and acute hepatopancreatic necrosis, red leg disease, gill rot, shell ulceration, and soft shell disease in shrimp. Consuming contaminated raw seafood can cause acute gastroenteritis and diarrhea in humans. Epidemiological studies indicate that Vibrio parahaemolyticus is, besides Vibrio cholerae, the most commonly reported pathogenic Vibrio to humans in clinical practice.

[0003] Currently, antibiotic therapy remains the primary means of controlling bacterial diseases in Litopenaeus vannamei, playing a crucial role in vibriosis control. However, the selective pressure caused by the overuse of antibiotics has accelerated the emergence and spread of drug-resistant strains, significantly reducing the effectiveness of drug control and posing a serious threat to the ecological environment and food safety. According to current regulations, only 13 antibiotic preparations are permitted for use in aquaculture, including thiamphenicol powder, florfenicol series preparations, and flumethylquine powder. It is noteworthy that due to the increasingly serious problem of drug resistance, drugs such as tetracycline and oxytetracycline, originally used to treat acute hepatopancreatic necrosis disease (AHPND), have been restricted in their use.

[0004] Bacteriophages, a class of viruses that specifically infect bacteria, are widely distributed in nature. Among them, schizophages can efficiently infect host bacteria, rapidly multiplying and ultimately causing bacterial lysis and death. This characteristic makes them significant in the field of food microbiology control. Since 2006, the first phage preparation (LiestShied) for controlling Listeria monocytogenes in meat has been widely used. TM Since receiving FDA approval, a variety of phage products have been approved for market launch, providing new ideas for solving the problem of antibiotic abuse.

[0005] Compared with traditional antibiotics, bacteriophages have unique advantages: their bactericidal effect is highly strain-specific, allowing for precise targeting of pathogens; once the host bacteria are eliminated, the bacteriophage loses its activity and does not remain in the host, posing no risk to aquatic ecosystems or human health. These characteristics make bacteriophages a promising candidate for application in the field of aquatic pathogen control. Summary of the Invention

[0006] The purpose of this invention is to provide a Vibrio parahaemolyticus phage LVPP58, the preservation number of which is CCTCC NO: M2025322.

[0007] Another object of the present invention is to provide a composition comprising Vibrio parahaemolyticus phage LVPP58 and florfenicol.

[0008] The final object of the present invention is to provide the use of the above-described bacteriophage or a combination thereof in the preparation of treatments or preventions of diseases caused by Vibrio parahaemolyticus infection.

[0009] To achieve the above objectives, the present invention adopts the following technical measures:

[0010] The applicant collected samples of aquaculture wastewater or pond water and screened different types of Vibrio parahaemolyticus strains. Finally, a commercially valuable Vibrio parahaemolyticus phage was selected. This phage was deposited at the China Center for Type Culture Collection (CCTCC) on February 28, 2025. The deposit address is: Wuhan University, Wuhan, China; the accession number is: CCTCC NO:M 2025322; and the classification name is: Vibrio parahaemolyticus phage LVPP58.

[0011] Bacteriophage LVPP58 is tadpole-shaped, with a head measuring 60-70 nm. It has a symmetrical icosahedral head with tiny spiky protrusions. The tail has relatively obvious segmentation features, is long and curled, and can reach 200 nm when fully extended.

[0012] The scope of protection of this invention also includes:

[0013] A compound comprising Vibrio parahaemolyticus phage with accession number CCTCC NO: M 2025322.

[0014] Preferably, the compound described above also includes florfenicol.

[0015] In the above-described compound, preferably, the ratio of Vibrio parahaemolyticus phage to florfenicol is 8.6 × 10⁻⁶. 6 PFU / mL: 0.125 μg / mL.

[0016] The above-mentioned bacteriophages or their compounds are used in the preparation of drugs for the treatment or prevention of Vibrio parahaemolyticus and / or Vibrio alginolyticus infections.

[0017] The application of the above-mentioned bacteriophages or their complexes in the preparation of antibacterial agents against Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0018] The above-mentioned bacteriophages or their complexes are used in the non-therapeutic in vitro inhibition of Vibrio parahaemolyticus and / or Vibrio alginolyticus.

[0019] The above-mentioned bacteriophages or their compounds are used in the preparation of drugs for the treatment or prevention of vibriosis in aquatic animals.

[0020] In the above-described applications, the preferred aquatic animal is the shrimp.

[0021] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0022] (1) Bacteriophage LVPP58 exhibits a broad host spectrum, capable of efficiently lysing 11 different types of Vibrio parahaemolyticus (including 8 ST forms), while also demonstrating strong bactericidal activity against 4 Vibrio alginolyticus strains. This characteristic breaks through the traditional limitation of strong host specificity of bacteriophages, significantly broadening its potential application range.

[0023] (2) Bacteriophage LVPP58 survives in a wide pH range of 3-11 (>10) 7 It maintains stable pyrolytic activity under conditions of PFU / mL and 30-60℃, indicating good acid and alkali tolerance and thermal stability. This characteristic makes it more adaptable and operable in practical production applications.

[0024] (3) The combination of bacteriophage LVPP58 and antibiotic florfenicol can produce a significant synergistic antibacterial effect. Experimental data showed that the fractional inhibitory concentration index (FIC) within 4 hours of the combination was 0.375, confirming its synergistic effect and providing a theoretical basis for the combination drug strategy.

[0025] (4) Vibrio parahaemolyticus phage LVPP58 has the characteristics of short latency period (<30 min) and large average lysis amount (>100 phages / cell). Attached Figure Description

[0026] Figure 1 Photograph of phage plaques of Vibrio parahaemolyticus phage LVPP58 on a double-layer agar plate.

[0027] Figure 2 This is an electron micrograph of Vibrio parahaemolyticus phage LVPP58.

[0028] Figure 3 This is a circumscribed map of the genome of bacteriophage LVPP58.

[0029] Figure 4 This is a one-step growth curve of Vibrio parahaemolyticus phage LVPP58.

[0030] Figure 5The lysis curves of LVPP58 against Vibrio parahaemolyticus LJVP6 are shown at different multiplicity of infection.

[0031] Figure 6 The results show the temperature tolerance of bacteriophage LVPP58.

[0032] Figure 7 Results for pH tolerance of bacteriophage LVPP58.

[0033] Figure 8 The results show the antibacterial effect of bacteriophage LVPP58 combined with different concentrations of florfenicol.

[0034] Figure 9 The results show the antibacterial effects of bacteriophage LVPP58 combined with 1 / 2MIC, 1 / 4MIC and 1 / 8MIC florfenicol.

[0035] Figure 10 Results of challenge with Vibrio parahaemolyticus LJVP6 in Litopenaeus vannamei.

[0036] Figure 11 The results of bacteriophage LVPP58 in controlling vibrio infection in Litopenaeus vannamei.

[0037] Figure 12 The results of using bacteriophage LVPP58 in combination with antibiotics to control vibrio infection in Litopenaeus vannamei;

[0038] LVPP58 was used in combination with florfenicol in the 1 / 2MIC, 1 / 4MIC and 1 / 8MIC ranges. Detailed Implementation

[0039] The present invention will be further illustrated below with reference to embodiments, but the scope of protection of the present invention is not limited to the scope described in the embodiments. Unless otherwise specified, the technical solutions described in the present invention are conventional techniques; the reagents or materials described, unless otherwise specified, are all from commercial sources.

[0040] Example 1:

[0041] Isolation and screening methods for Vibrio parahaemolyticus phage LVPP58

[0042] Collect 10 mL of aquaculture wastewater or pond water sample, centrifuge at 10000×g for 10 min at 4℃, take the supernatant and filter it through filter paper to remove large particulate impurities, then filter it through a 0.22 μm filter membrane, and store the filtrate at 4℃ for later use.

[0043] Remove Vibrio parahaemolyticus strains (host strains used are shown in Table 1) from a -80℃ freezer and streak them onto LA solid medium containing 3% NaCl (components: 1% tryptone, 0.5% yeast extract, 1.5% agar, 3% NaCl), and incubate at 37℃ for 8-12 hours. Pick a single colony and inoculate it into 5 mL of LB liquid medium (components: 1% tryptone, 0.5% yeast extract, 3% NaCl), and incubate at 37℃ with shaking until the logarithmic growth phase (OD200). 600 The value is 0.5-0.6).

[0044] Table 1 shows the MLST typing of the host Vibrio parahaemolyticus used.

[0045]

[0046] Mix the bacterial culture, filtrate, and culture medium at a ratio of 1:2:4 (2.5 mL host bacterial culture, 5 mL filtrate, and 10 mL LB medium), and incubate at 37°C and 200 rpm with shaking for 8–12 h. After centrifugation (10000 × g, 10 min, 4°C), collect the supernatant, filter through a 0.22 μm filter membrane, and collect the filtrate.

[0047] To verify the isolation of bacteriophages using the spot method, the specific steps are as follows: Mix 100 μL of host bacterial culture in the logarithmic growth phase with 3.8 mL of 0.7% LB semi-solid medium, pour the mixture onto an LA solid plate, and allow it to solidify. Spot 5 μL of the filtrate onto the surface of the upper agar layer, allow it to air dry, and then incubate at 37°C for 8-12 hours. Observe the formation of bacteriophage plaques; the appearance of clear plaques indicates that bacteriophages have been isolated using the above experimental steps.

[0048] Purification and proliferation of bacteriophages

[0049] After successfully isolating the bacteriophage, it needs to be purified. The purification steps are as follows: Pick a single plaque with a sterile pipette tip, add it to 100 μL of host bacterial culture and 10 mL of LB liquid medium, and incubate at 37°C with shaking for 8-12 h. After incubation, centrifuge (10000×g, 10 min, 4°C), filter the supernatant through a 0.22 μm filter membrane, and collect the filtrate to obtain the proliferated bacteriophage stock solution.

[0050] Perform 10-fold serial dilutions of the phage stock solution. Mix 100 μL of the appropriate dilution with 100 μL of the host bacterial culture and 3.8 mL of LB semi-solid medium. Pour the mixture onto LA plates, allow it to stand and cool, and then incubate at 37°C upside down for 8-12 hours. Repeat the amplification of single phage plaques 3-5 times to ensure phage purity.

[0051] Phage titer determination

[0052] The titer of a bacteriophage, also known as the plaque-forming unit (PFU) or titer, represents the number of bacteriophages present per milliliter of liquid. This experiment determined the titer using the double-layer plate method. Specifically, the purified bacteriophage stock solution was serially diluted 10-fold. 100 μL of each appropriate dilution was mixed with 100 μL of host bacterial culture and 3.8 mL of LB semi-solid medium, poured onto LA plates, and incubated overnight at 37°C with the plates inverted. Each dilution was replicated in triplicate. The titer calculation formula is as follows:

[0053] Phage titer (PFU / mL) = Number of phage plaques × Dilution factor × 10

[0054] Table 2 shows some of the isolated Vibrio parahaemolyticus phages.

[0055] Table 2 shows partial screening information for Vibrio parahaemolyticus phages.

[0056]

[0057] Preservation of bacteriophages

[0058] To facilitate subsequent research, the purified phage was preserved using the glycerol preservation method, which involved mixing 300 μL of 80% glycerol with 900 μL of phage solution and freezing it at -80°C.

[0059] Example 2:

[0060] Phage host profile determination and acquisition of Vibrio parahaemolyticus phage LVPP58:

[0061] The host spectrum was determined using a serial dilution drop method, as follows: Phage stock solutions were serially diluted 10-fold for later use. Fifteen different serotypes of *Vibrio parahaemolyticus* and seventeen different serotypes of *Vibrio alginolyticus* (Table 3) were inoculated into 5 mL of LB medium and incubated at 37°C until the logarithmic development phase. 100 μL of the test bacterial solution was mixed with 3.8 mL of 0.7% LB semi-solid medium and quickly poured onto LA solid plates, allowing them to solidify. 5 μL of each serially diluted phage solution was spotted onto the surface of the upper agar plate, air-dried, and incubated upside down at 37°C overnight. The next day, plaque formation was observed, and the host spectrum was statistically analyzed (results shown in Table 3).

[0062] Table 3 shows a partial spectrum of phage hosts selected for screening.

[0063]

[0064] Note: "+" indicates that the bacteriophage can use this strain of bacteria as a host, and "-" indicates that the bacteriophage cannot use this strain of bacteria as a host.

[0065] The phage host spectrum results show that Vibrio parahaemolyticus phage LVPP58 can use 11 Vibrio parahaemolyticus strains (covering 8 ST types) and 4 Vibrio alginolyticus strains as hosts, exhibiting a highly significant broad spectrum. Based on this, a novel broad-spectrum Vibrio parahaemolyticus phage, LVPP58, was successfully screened, and will be referred to as LVPP58 in this invention.

[0066] The applicant deposited the following on February 28, 2025, at the China Center for Type Culture Collection (CCTCC), classified and named as Vibrio parahaemolyticus phage LVPP58, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2025322.

[0067] Example 3:

[0068] Morphological characterization of Vibrio parahaemolyticus phage LVPP58

[0069] Morphological observation of LVPP58 on double-layer plates: 100 μL of host bacterial culture (LJVP6), 100 μL of appropriately diluted phage fluid, and 3.8 mL of 0.7% LB semi-solid medium were mixed and quickly poured onto LA plates. After solidification, the plates were incubated upside down at 37°C overnight. The morphology of the phage plaques was observed and recorded the next day. Figure 1 ).

[0070] Morphage morphology observation under transmission electron microscopy: First, the titer of the bacteriophage was increased to 10. 9 PFU / mL or higher. After rinsing the copper mesh with sterile water, immerse it in the phage solution on ice for 5 min. Blot dry with filter paper, negatively stain with 2% phosphotungstic acid (PTA) for 10 min, and allow it to air dry in a cool place before observation using a transmission electron microscope at an accelerating voltage of 75 kV. Morphological measurements were performed using ImageJ software based on ≥20 intact phage particles.

[0071] The results are as follows Figure 2 As shown, under an electron microscope, bacteriophage LVPP58 was found to be tadpole-shaped, with a head measuring 60-70 nm, featuring a symmetrical icosahedral head and tiny spiny protrusions. The tail exhibited distinct segmentation, was long and curled, and could reach 200 nm when fully extended. Based on the International Classification of Viruses (ICTV), it is preliminarily classified into the family Longtailed Phages, order Caudate Phages.

[0072] Example 4:

[0073] Genome analysis of Vibrio parahaemolyticus phage LVPP58

[0074] Genome circle diagram as follows Figure 3As shown, Vibrio parahaemolyticus phage LVPP58 uses double-stranded DNA as its genetic material, with a genome size of 81297 bp and a G+C content of 46%. Open reading frame (ORF) function prediction and analysis revealed 117 ORFs, of which 47 had clearly defined functions and were annotated as structural modules, cleavage modules, DNA replication modules, and nucleic acid metabolism modules, respectively. The remaining 70 ORFs were hypothetical proteins with unknown functions.

[0075] Example 5:

[0076] One-step growth curve determination of Vibrio parahaemolyticus phage LVPP58

[0077] Prepare host bacterial culture in the logarithmic phase and dilute the culture concentration to 10. 6 CFU / mL. Mix 10 at the optimal multiple of infection (MOI = 0.1). 5 PFU / mL phage and 10 6 500 μL of host bacterial culture (CFU / mL) was mixed thoroughly using a vortex mixer. After adsorption at 37°C according to the adsorption rate determination, the culture was centrifuged at 4°C and 8000 rpm for 2 min, the supernatant was discarded, and the sample was washed twice with PBS buffer. The supernatant was then discarded again after centrifugation. Finally, the sample was resuspended in 10 mL of LB medium. The sample was immediately placed in a shaker at 37°C and the timer was started simultaneously. For the first 20 min, 300 μL samples were taken every 5 min, and then every 10 min. After centrifugation at 4°C and 8000 rpm for 30 s, 100 μL of the supernatant was immediately transferred to 900 μL of LB medium for serial dilution. The phage titer was determined using the double-layer plate method after selecting an appropriate dilution gradient.

[0078] The final result is as follows Figure 4 As shown, the phages began to multiply rapidly about 20 minutes after inoculation, and the titer exceeded 1×10⁻⁶ about 1 hour after inoculation. 6 The phage titer initially increased by one order of magnitude every 20-40 minutes, reaching its peak value (approximately 10 PFU / mL) about 4 hours after inoculation. 9 (Approximately PFU / mL). This indicates that Vibrio parahaemolyticus phage LVPP58 has the characteristics of a short latency period (<30 min) and a large average lysis rate (>100 phages / cell).

[0079] Example 6:

[0080] Determination of lysis curve of Vibrio parahaemolyticus phage LVPP58

[0081] Culture Vibrio parahaemolyticus bacterial suspension to increase its OD 600 The value is between 0.5 and 0.6, at which point the bacterial concentration is 10. 8CFU / mL, serially diluted to a bacterial concentration of 10. 6 CFU / mL was prepared for use. The LVPP58 phage stock solution was serially diluted to different concentrations. 100 μL of phage solution at different dilutions were taken and diluted according to MOIs of 100, 10, 1, 0.1, 0.01, and 0.001, respectively, with a concentration of 10⁻⁶ CFU / mL. 6 Mix the bacterial suspension at CFU / mL. A blank control group was set up: 200 μL of LB medium was added; a positive control group was set up: 100 μL of logarithmic-phase host bacterial suspension and 100 μL of LB medium were added. Microplate reader parameters were set as follows: λ = 600 nm, temperature set to 37.0℃. OD values ​​were measured every 1 hour for a total of 12 hours.

[0082] The final result is as follows Figure 5 As shown, when MOI = 100, in the initial stage of the experiment (0-5h), OD 600 The OD value increased slowly, indicating that the phage's inhibitory effect on the growth of the bacteriophage was most significant at this time, while in the middle of the experiment (5-10h), the OD value increased more slowly. 600 The rapid increase in value may be due to an excessive amount of bacteriophages caused by an excessively high MOI, which affects the normal infection process. Subsequently, at the end of the experiment (10-15h), the bacterial growth rate slowed down again, indicating that the growth of Vibrio parahaemolyticus was inhibited again at this time.

[0083] When MOI = 10, 1, 0.1, and 0.01, the lysis curves initially rise, then plateau or decline at different time points, before continuing to rise. Taking MOI = 0.1 as an example, the curve's upward trend slows after approximately 4 hours, reaching a relatively stable phase, before continuing to rise. This indicates that bacteriophages have a certain lysis effect on bacteria at a given MOI, reducing the number of bacteria in the bacterial culture and decreasing OD. 600 The value decreases, but over time, some bacteria may develop resistance, or bacteria not infected by bacteriophages may continue to grow, leading to an increase in OD. 600 The value rose again.

[0084] Example 7:

[0085] Temperature tolerance test of Vibrio parahaemolyticus phage LVPP58

[0086] The potency is approximately 10 8 500 μL of PFU / mL phage solution was placed in constant temperature water baths at 40℃, 50℃, 60℃, 70℃, and 80℃. 100 μL of phage lysate was taken at 30 min and 60 min respectively. After equilibration at room temperature, the titer was determined using the bilayer plate method. The final results are shown in Table 4. Figure 6 As shown, the potency of LVPP58 remains stable (>10) within the temperature range of 30-60℃. 8The PFU / mL indicates that the phage has strong temperature tolerance.

[0087] Table 4 Temperature tolerance of bacteriophage LVPP58

[0088]

[0089] Example 8:

[0090] pH tolerance assay of Vibrio parahaemolyticus phage LVPP58

[0091] 100 μL of phage solution was mixed with 900 μL of LB medium at pH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13, respectively, and incubated in a 37°C water bath for 2 hours. The phage titer was then determined using the double-layer plate method. The final results are shown in Table 5. Figure 7 As shown, the potency of LVPP58 remains stable within the pH range of 3-11 (>10). 7 The PFU / mL value indicates that the phage has strong pH tolerance.

[0092] Table 5 pH tolerance of bacteriophage LVPP58

[0093]

[0094] Example 9:

[0095] Optimal Multiple of Infection Determination for Vibrio parahaemolyticus phage LVPP58

[0096] The multiplicity of infection (MOI) is the ratio of the number of bacteriophages added at the time of initial infection to the number of host bacteria; it is also called the infection multiple.

[0097] Phage fluid and 10 6 100 μL of host bacterial culture at CFU / mL was added to 800 μL of LB medium and mixed well. The mixture was incubated at 37℃ and 100 rpm for 3.5 h, then centrifuged at 4℃ and 10000 rpm for 10 min. 100 μL of the supernatant was collected and serially diluted 10-fold with LB medium. The phage titer was then determined using the double-layer plate method. The highest multiplicity of infection (MOI) was the optimal MOI for this phage. The final results are shown in Table 6. The optimal MOI for Vibrio parahaemolyticus phage LVPP58 was 0.1, indicating that this phage has highly efficient infective properties.

[0098] Table 6 Optimal Multiplicity of Infection for Bacteriophage LVPP58

[0099] MOI Potency (PFU / mL) 0.001 <![CDATA[3.00×10 8 ]]> 0.001 <![CDATA[9.60×10 8 ]]> 0.1 <![CDATA[3.50×10 9 ]]> 1 <![CDATA[9.70×10 7 ]]> 10 <![CDATA[1.14×10 7 ]]> 100 <![CDATA[3.10×10 6 ]]> 1000 <![CDATA[2.60×10 6 ]]>

[0100] Example 10:

[0101] Evaluation of the combined antibacterial effect of Vibrio parahaemolyticus phage LVPP58 and florfenicol (FFC)

[0102] The minimum inhibitory concentration (MIC) of LVPP58 was determined using the standardized microbroth dilution method. Vibrio parahaemolyticus LJVP6 was cultured to the logarithmic growth phase (OD = 0.7, approximately 10⁻⁶). 9 CFU / mL), diluted to 1.0 × 10⁻⁶ with LB medium. 6 CFU / mL. Preparation 1.37 × 10 8 ~1.1×10 6 PFU / mL phage serial dilution buffer (2-fold serial dilution). Add the bacterial culture and phage dilution buffer to a 96-well microtiter plate (Grenier-Bio 655-185) to a final volume of 0.2 mL per well. Initial optical density (OD) 600 The value was controlled at around 0.1. The culture was carried out at 37℃ for 3 hours, and the optical density (OD) was measured every 30 minutes using a BioTek Cytation 3 microplate reader. 600 Each group was repeated 3 times. The lowest drug concentration at which bacterial growth inhibition ≥50% was defined as the half-maximal inhibitory concentration (IC50), while the lowest drug concentration at which bacterial growth inhibition ≥90% was defined as the MIC (IC90). To avoid error, the concentration at which optical density (OD) was measured was considered to be... 600 When the optical density (OD) is 0.2, the liquid is turbid. 600 The lowest concentration of bacteriophage used when the concentration is less than 0.2 is considered the minimum inhibitory concentration (MIC) for combined use.

[0103] The MIC of florfenicol (FCC) single drug was also determined using the microbroth dilution method. A stock solution of florfenicol was prepared at a concentration of 256 mg / mL and diluted to 0.063 μg / mL with LB medium. 100 μL of the 0.063 μg / mL solution was added to wells 1-2 of a 96-well plate, and 100 μL of LB medium was added to wells 2-12. Serial dilutions were performed from well 2 to well 12. Vibrio parahaemolyticus LJVP6 was cultured to the logarithmic development phase and adjusted to 1×10⁶ ppm with LB medium. 6 CFU / mL, 50 μL of bacterial culture was added to each well, and 50 μL of LB medium was added to the 12th well. The 12th well served as a positive control (LJVP6 + LB medium), and a separate well served as a negative control (LB medium). The 96-well plate was incubated at 37°C for 20 h. Optical density (OD) was measured every 30 min using a BioTek Cytation3. 600 Repeat 3 times for each group.

[0104] The procedure for the combined phage and sub-inhibitory concentration (MIC) FCC assay is as follows: Prepare the FCC minimum inhibitory concentration (MIC) solution. Add 100 μL of LB medium to each well of a 96-well plate. Dilute the antibiotic to 1 / 2 MIC, 1 / 4 MIC, and 1 / 8 MIC using a serial dilution method. Add 50 μL of LJVP6 bacterial culture (1×10⁻⁶) cultured to the logarithmic growth phase to each well. 6 CFU / mL) and phage LVPP58 dilution (potency 10) 7 PFU / mL). The 96-well plate was incubated at 37°C for 20 h. Optical density (OD) was measured every 30 min using a BioTek Cytation3. 600 Each group was repeated 3 times. Results are as follows: Figure 9 As shown.

[0105] Construct an 8×8 concentration matrix. Group A (vertical axis) is treated with florfenicol (FFC) at final concentrations of 4–1 / 32 μg / mL, added sequentially from top to bottom. Group B (horizontal axis) is treated with florfenicol (FFC) at a potency of 1.37 × 10⁻⁶ μg / mL, added sequentially from left to right. 8 ~1.1×10 6 Phage at PFU / mL. Vibrio parahaemolyticus cultured to the logarithmic growth phase was diluted to 1.0 × 10⁻⁶. 6 CFU / mL was added to a 96-well plate, resulting in a final volume of 0.2 mL per well. The initial optical density (OD) was... 600 The value was controlled at around 0.1. The cells were incubated at 37℃ for 3 hours, and the optical density (OD) was measured every 30 minutes. 600 To avoid errors, optical density (OD) 600 The lowest concentration at which bacteriophage and florfenicol are used in combination is considered the minimum inhibitory concentration (MIC) for combined use, provided that the MIC is below 0.2. At this point, the florfenicol concentration is MIC (for group A combined use), and the bacteriophage concentration is MIC (for group B combined use).

[0106] Calculation of FIC index: FIC index = MIC (FCC in combination) / MIC (FCC alone) + MIC (phage in combination) / MIC (phage alone)

[0107] FIC index interpretation criteria: When the FIC index is less than 0.5, the two drugs have a synergistic effect; when the FIC index is between 0.5 and 1, the two drugs have an additive effect; when the FIC index is greater than 1 and less than 2, the two drugs have no effect; when the FIC index is greater than 2, the two drugs have an antagonistic effect.

[0108] Growth curves when used in combination: Figure 8 As shown in the figure, the antibacterial effect of FFC and LVPP58 alone is not as significant as that of the combination of the two.

[0109] As shown in Table 7, the calculated FIC of Vibrio parahaemolyticus phage LVPP58 combined with florfenicol within 4 hours was 0.375, indicating a strong synergistic effect.

[0110] This experiment confirms that the combined use of bacteriophage LVPP58 and florfenicol can produce a significant systemic antibacterial effect, providing an important basis for the formulation of clinical combination drug regimens.

[0111] Table 7. Minimum inhibitory concentration of Vibrio parahaemolyticus phage LVPP58 combined with florfenicol within 4 hours.

[0112]

[0113] Example 11:

[0114] Establishment of an infection model in Litopenaeus vannamei and LD 50 Measurement

[0115] One hundred and five Litopenaeus vannamei shrimp (7±1 cm in length) were temporarily held for one week to confirm they were free of pathogen infection. They were then randomly divided into seven groups (six experimental groups and one control group), with 15 shrimp in each group. The seven groups of shrimp were placed in 5L PVC tanks (each tank containing 3L of sterile seawater with independent circulation). The water temperature was maintained at 28±1℃, dissolved oxygen above 5 mg / L, and pH 7.5-8.5. The shrimp were fasted for 24 hours prior to viral challenge. Vibrio parahaemolyticus LJVP6 was cultured overnight, centrifuged, and then serially diluted with sterile physiological saline to an infectious concentration of 1.0 × 10⁻⁶. 9 1.0×10 8 1.0×10 7 1.0×10 6 1.0×10 5 1.0×10 4 CFU / mL. Immersion infection was employed; the experimental groups were immersed in their respective bacterial solutions for 1 hour (the control group was treated with sterile saline), and then transferred to an equal volume of sterile water. During the experiment, the water was not changed, and the animals were kept hungry, maintaining water quality parameters such as dissolved oxygen and temperature consistent with the temporary rearing period. Mortality and typical symptoms were observed and recorded at 6h, 12h, 24h, 48h, and 72h. The 72h LD50 was calculated after 3 days. 50 The result is as follows Figure 10 As shown. The final calculation result is LD. 50 =1.47×10 5 CFU / mL.

[0116] Example 12:

[0117] Evaluation of the control efficacy of Vibrio parahaemolyticus phage LVPP58 against vibrio infection in Litopenaeus vannamei.

[0118] Healthy Litopenaeus vannamei (same as in Example 11) were divided into three groups: a prevention group treated with bacteriophage followed by pathogen treatment, a treatment group treated with pathogen treatment followed by bacteriophage treatment, and a control group treated only with pathogen treatment. Each group consisted of 15 shrimp. The culture system and water environment parameters were the same as in Example 11. Prevention and treatment were conducted at a bacteriophage concentration to Vibrio parahaemolyticus bacterial solution concentration ratio of 1:1 (MOI = 1), with a challenge concentration of 1.47 × 10⁻⁶. 7 CFU / mL (100 times LD50) 50 The method of attack was immersion infection.

[0119] The prevention group was treated by placing Litopenaeus vannamei in a final concentration of 1.47 × 10⁻⁶. 7 After soaking in phage fluid at PFU / mL for 1 hour, the phage was transferred to a bacterial culture with a concentration of 1.47 × 10⁻⁶ PFU / mL. 7 CFU / mL (100LD) 50 Soak in an equal volume of water for 1 hour, then transfer to an equal volume of sterile seawater.

[0120] The treatment group was prepared by placing Litopenaeus vannamei at a final concentration of 1.47 × 10⁻⁶. 7 After soaking in bacterial suspension at CFU / mL for 1 hour, the culture was transferred to a phage concentration of 1.47 × 10⁻⁶. 7 After soaking in an equal volume of water containing PFU / mL for 1 hour, the sample was transferred to an equal volume of sterile seawater.

[0121] The control group used 100LD 50 The shrimp were challenged by immersion in a bacterial solution of a specific concentration for 1 hour, and then transferred to an equal volume of sterile seawater. During the challenge period, the water was not changed, and the shrimp were not fed. Dissolved oxygen, temperature, and other water quality parameters were maintained consistent with those during the temporary holding period. The observation period was 72 hours. The number of dead Litopenaeus vannamei shrimp was counted daily, and the dead shrimp were removed. The relative protection rate (RPS) was calculated using the following formula:

[0122] Relative immune protection rate (RPS) = (1 - mortality rate of prevention and treatment group / mortality rate of control group) × 100%.

[0123] Survival rate curves were plotted using Prism 10.0 software.

[0124] The results are as follows Figure 11As shown, the survival rate of Litopenaeus vannamei in the control group dropped sharply shortly after infection, while both phage treatment and prevention significantly improved the survival rate, with RPS of 46.2% in the treatment group and 53.8% in the prevention group. This indicates that phage LVPP58 has a good preventive and control effect against Vibrio parahaemolyticus infection. The survival rate of the prevention group was higher than that of the treatment group throughout the experiment, demonstrating that phage prevention treatment can more effectively protect Litopenaeus vannamei and reduce its mortality rate. This example provides a new strategy for the early biocontrol of Vibrio parahaemolyticus in shrimp farming.

[0125] Example 13:

[0126] Evaluation of the efficacy of Vibrio parahaemolyticus phage LVPP58 combined with florfenicol in the prevention and control of vibrio infection in Litopenaeus vannamei.

[0127] Healthy Litopenaeus vannamei (same as in Example 11) were divided into three groups: a prevention group, a treatment group, and a control group, with 15 shrimp in each group. The culture conditions for each group were the same as in Example 11. In both the prevention and treatment groups, the ratio of bacteriophage to Vibrio parahaemolyticus bacterial suspension was 1:1 (MOI = 1), and the challenge concentration was 1.47 × 10⁻⁶. 7 CFU / mL (100LD) 50 The challenge method was immersion infection. The prevention group was treated by first adding purified bacteriophage and florfenicol solution to the water tank until the final concentration was 1.47 × 10⁻⁶. 7 After soaking in PFU / mL and 1 / 2 MIC for 1 hour, the solution was transferred to a bacterial culture concentration of 1.47 × 10⁻⁶. 7 CFU / mL (100LD) 50 Soak in an equal volume of water for 1 hour, then transfer to an equal volume of sterile seawater.

[0128] The treatment group was prepared by placing Litopenaeus vannamei in a phage culture with a final FCC concentration of 1.47 × 10⁻⁶. 7 After soaking in water with PFU / mL and 1 / 2 MIC for 1 hour, the culture was transferred to a solution with a bacterial concentration of 1.47 × 10⁻⁶. 7 After soaking in an equal volume of water at CFU / mL for 1 hour, the samples were transferred to an equal volume of sterile seawater. The control group used 100 times LD50. 50 The bacterial concentration was 1.47 × 10⁻⁶. 7 The shrimp were challenged by immersion in a solution of CFU / mL, and after 1 hour, they were transferred to an equal volume of sterile seawater. During the challenge period, the water was not changed, and the shrimp were not fed. Dissolved oxygen, temperature, and other water quality parameters were maintained consistent with those during the temporary holding period. The observation period was 72 hours. The number of dead shrimp was counted daily, and the dead individuals were removed. The relative protection rate (RPS) was calculated according to the method in Example 12, and survival curves were plotted.

[0129] The results are as follows Figure 12As shown, the combined application of bacteriophage LVPP58 and florfenicol significantly improved the survival rate of Litopenaeus vannamei infected with Vibrio parahaemolyticus, whether used for prevention or treatment. The recurrence-free survival (RPS) of the prevention and treatment groups were 76.9% and 69.2%, respectively, with the combined use being superior to florfenicol alone. Furthermore, the preventive treatment was more effective than the treatment. This combined strategy can serve as an effective disease control measure, reducing economic losses caused by Vibrio parahaemolyticus infection.

Claims

1. An isolated bacteriophage (phage) of Vibrio parahaemolyticus, LVPP58, characterized by: Vibrio parahaemolyticus The preservation number of the strain is CCTCC NO: M2025322. ​ 2. A complex preparation comprising the Vibrio parahaemolyticus bacteriophage with the preservation number of CCTCC NO: M2025322.

3. The complex preparation according to claim 2, which comprises florfenicol.

4. The combination of claim 3, wherein the ratio of Vibrio parahaemolyticus bacteriophage to florfenicol in said combination is 8.6 x 10 6 PFU / mL: 0.125 μg.

5. Use of the bacteriophage according to claim 1 or the complex preparation according to claim 2 in the preparation of a medicament for treating or preventing Vibrio parahaemolyticus and / or Vibrio alginolyticus infection.

6. Use of the bacteriophage according to claim 1 or the complex preparation according to claim 2 in the preparation of a Vibrio parahaemolyticus and / or Vibrio alginolyticus bacteriostatic agent.

7. Use of the bacteriophage according to claim 1 or the complex preparation according to claim 2 in the non-therapeutic in vitro inhibition of Vibrio parahaemolyticus and / or Vibrio alginolyticus.

8. Use of the bacteriophage according to claim 1 or the complex preparation according to claim 2 in the preparation of a medicament for treating or preventing Vibrio parahaemolyticus and / or Vibrio alginolyticus infection in aquatic animals.

9. The use according to claim 8, wherein the aquatic animals are prawns.

Images