PhLNJZ2, a broad-spectrum short-tailed bacteriophage targeting multidrug-resistant Vibrio, and its applications.

Abstract

This invention discloses a broad-spectrum short-tailed bacteriophage, PhLNJZ2, targeting multidrug-resistant Vibrio and its applications, belonging to the field of microbial technology. The bacteriophage PhLNJZ2 was deposited on October 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO:M 20252251, and classified as follows: Vibrio Phage PhLNJZ2. This bacteriophage was screened from a clinical isolate of a multidrug-resistant Vibrio alginolyticus resistant to more than seven antibiotics. It exhibits highly efficient and broad-spectrum lytic activity against three major aquatic pathogenic Vibrio species: Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi, with a total lysis rate of 81.25%. This invention also provides compositions containing this bacteriophage, feed additives, and their application in the preparation of drugs or environmental disinfectants for the prevention and control of multidrug-resistant and mixed vibrio infections in aquatic organisms.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a broad-spectrum, highly virulent short-tailed bacteriophage PhLNJZ2 isolated from multidrug-resistant Vibrio alginolyticus and its application in the preparation of formulations for the prevention or treatment of vibriosis infections caused by multidrug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, and / or Vibrio harveyi. Background Technology

[0002] Vibrio infection is a major threat to the healthy development of aquaculture, including shrimp, sea cucumber, and fish farming. The main pathogens include Vibrio alginolyticus (Villus alginolyticus). Vibrio alginolyticus ), Vibrio parahaemolyticus ( V. parahaemolyticus ) and Vibrio harveyi ( V. harveyi In actual aquaculture environments, mixed infections of multiple Vibrio species are common. Furthermore, the long-term and widespread use of antibiotics has led to the emergence and prevalence of multidrug-resistant (MDR) Vibrio strains, reducing the effectiveness of traditional chemical treatments, increasing aquaculture costs, and posing risks of drug residues and environmental hazards. Therefore, developing novel, environmentally friendly biological control methods that effectively combat MDR Vibrio has become an urgent need in the aquaculture industry.

[0003] Phage therapy, as an alternative strategy capable of specifically lysing bacteria, shows promising application prospects due to its advantages such as high specificity, self-replication ability, good environmental compatibility, and low likelihood of inducing broad-spectrum drug resistance in bacteria. Currently, several phages targeting pathogenic Vibrio in aquatic organisms have been isolated and reported, providing potential resources for the biocontrol of vibrio infections.

[0004] For example, certain Vibrio alginolyticus phages are disclosed in the prior art, which have certain application potential in water disinfection or feed additives. However, when dealing with the complex situation of multidrug-resistant Vibrio infections and mixed infections of multiple Vibrio species that are currently quite challenging in aquaculture, existing phage resources still have the following limitations: Insufficient targeting of multidrug-resistant strains: Most reported bacteriophages were isolated from routinely sensitive strains or environmental samples, rather than specifically targeted at clinically resistant multidrug-resistant Vibrio. Therefore, the lysis efficiency and application effectiveness of these bacteriophages against increasingly prevalent multidrug-resistant strains remain uncertain.

[0005] The matching degree between broad-spectrum lysis capability and actual mixed infection scenarios needs improvement: Although some bacteriophages have lysis activity against multiple strains within a single Vibrio species, their "true broad-spectrum" capability across species (such as simultaneously and efficiently lysing Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi) is limited. To address mixed infections, it is often necessary to combine multiple bacteriophage strains into "cocktail" formulations, which increases the complexity of the production process, the difficulty of quality control, and the cost of use.

[0006] The types of bacteriophage resources still need to be further enriched: Although some short-tailed Vibrio phages have been reported, short-tailed phages that specifically target multidrug-resistant clinical isolates and can efficiently cover the three main species of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi with high lytic activity are still relatively limited, and related resources need to be further explored.

[0007] In summary, there is still a need in this field for a novel phage that can target multidrug-resistant Vibrio, possesses efficient cross-species broad-spectrum lysis capabilities, exhibits stable biological characteristics, and is suitable for application in complex aquaculture environments. This would further enrich existing technological resources and provide core germplasm resources and solutions for the green control of drug-resistant Vibrio diseases in aquaculture. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a short-tailed bacteriophage, PhLNJZ2. This bacteriophage possesses the following superior characteristics: it is specifically isolated from multidrug-resistant Vibrio alginolyticus hosts; it exhibits highly efficient lytic activity against three major pathogenic bacteria: Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi; it has unique genomic characteristics and high safety; and it exhibits excellent environmental adaptability. The present invention also provides the application of this bacteriophage and its compositions in the preparation of formulations for the prevention or treatment of vibriosis infections caused by Vibrio in aquaculture.

[0009] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides a broad-spectrum short-tailed bacteriophage PhLNJZ2 targeting multidrug-resistant Vibrio. PhLNJZ2 was deposited on October 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20252251, and classified as follows: Vibrio phage PhLNJZ2.

[0010] Preferably, the genome of the phage PhLNJZ2 is a linear double-stranded DNA with a size of 32,736 bp and a GC content of 44.42%. More preferably, m4C and / or m6A methylation modifications are detected in its genome, a feature that may help enhance its environmental stability.

[0011] Preferably, the bacteriophage PhLNJZ2 exhibits lytic activity against test strains including *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Vibrio harveyi*. Experimental results show that the total lysis rate of this bacteriophage against 16 representative aquatic pathogenic Vibrio strains (covering the above three species, including standard strains and clinical isolates), including *Vibrio alginolyticus*, *Vibrio parahaemolyticus*, and *Vibrio harveyi*, is not less than 80%, preferably 81.25%; among them, the proportion of strains exhibiting strong lytic activity (+++) is not less than 56%, preferably 56.25%. This quantitative data strongly demonstrates that although bacteriophage PhLNJZ2 belongs to the family Short-tailed Bacteriophages, it possesses cross-species broad-spectrum lytic capabilities comparable to or even superior to some Long-tailed Bacteriophages, achieving unexpected technical effects.

[0012] In a second aspect, the present invention provides a phage composition comprising the phage PhLNJZ2 and a pharmaceutically, feed-grade, or environmentally acceptable carrier.

[0013] Preferably, the phage composition is a purified solution of phage PhLNJZ2, wherein the phage titer is not less than 1×10⁻⁶. 8 PFU / mL.

[0014] Thirdly, the present invention provides an aquatic feed additive comprising the bacteriophage PhLNJZ2 or the bacteriophage composition thereof.

[0015] Fourthly, the present invention provides the use of the bacteriophage PhLNJZ2, the bacteriophage composition, or the aquatic feed additive in the preparation of formulations for the prevention and / or treatment of vibriosis in aquatic animals. The vibriosis is preferably caused by multidrug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, and / or Vibrio harveyi, and / or a mixed infection of these multiple Vibrio species.

[0016] Preferably, the formulation includes pharmaceutical preparations for treatment or prevention, and environmental disinfectants for purifying aquaculture water. The dosage forms of the pharmaceutical preparations include, but are not limited to, solutions, powders, gels, granules, or lyophilized formulations.

[0017] Compared with the prior art, one of the technical solutions of the present invention has the following beneficial effects: (1) Provides a novel Vibrio phage resource with unique application value: This invention isolated and verified a new strain of Vibrio phage PhLNJZ2, a member of the Vibrio phage family, which exhibits highly efficient lysis activity against a variety of pathogenic Vibrio strains, including multidrug-resistant strains, adding a new resource with research and application value to the phage biocontrol library.

[0018] (2) Highly effective targeting of multidrug-resistant strains: This phage was specifically isolated to lyse multidrug-resistant Vibrio alginolyticus (resistant to 7 out of 14 tested antibiotics), providing a novel biological solution to the problem of bacterial resistance caused by antibiotic abuse, and has important clinical application value.

[0019] (3) Broad-spectrum lysis capability verified by experiments: While targeting drug-resistant bacteria, the single phage PhLNJZ2 achieved a total lysis rate of 81.25% against the test strains of three major pathogenic Vibrio species: Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi, which can effectively deal with mixed infections without the need for complex compounding.

[0020] (4) Excellent environmental adaptability and safety: Physophage PhLNJZ2 maintains high stability within the conventional aquaculture temperature range (4-37℃) and pH range (5.0-9.0), making it suitable for practical aquaculture applications. Whole genome analysis confirms that it does not carry virulence genes or drug resistance genes, and has high ecological safety.

[0021] (5) High lysis efficiency and significant control effect: This bacteriophage has a short incubation period (about 22 min) and a high burst rate (about 98 PFU / cell), which can rapidly lyse the host bacteria. In shrimp farming pond trials, the application of its preparation can rapidly and persistently reduce the concentration of pathogenic Vibrio in the water and significantly improve the shrimp survival rate (the shrimp survival rate reached 88%-92% after application), demonstrating excellent preventive and therapeutic effects.

[0022] (6) Possessing unique genomic methylation modifications and high environmental stability, it lays the biological foundation for long-term control. Whole-genome sequencing revealed that bacteriophage PhLNJZ2 has unique molecular characteristics (genome size 32,736 bp, GC content 44.42%) and contains m6A and m4C methylation modifications. This epigenetic feature may enhance the stability of its DNA in complex environments and its anti-host defense capabilities. Experiments have confirmed that it maintains high activity for a long time (24-hour retention rate >82%) under normal aquaculture conditions (4-37℃, pH 5.0-9.0). Its excellent stability upgrades it from a "short-acting disinfectant" to a "long-acting biological control agent" that can be used for ecological regulation of aquaculture waters, achieving a leap from treatment to prevention.

[0023] (7) Clear genomic background and guaranteed safety. Comprehensive bioinformatics analysis showed that the PhLNJZ2 phage genome does not carry any known bacterial virulence genes or antibiotic resistance genes, and its virulent phage characteristics also avoid the potential risks brought about by lysogenic conversion. Therefore, this phage has high ecological safety in environmental applications and meets the development requirements of green and healthy aquaculture. Attached Figure Description

[0024] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1 Photographs of phage plaques from PhLNJZ2 bacteriophage; Figure 2 Electron micrograph of bacteriophage PhLNJZ2; Figure 3 The results of temperature stability determination for bacteriophage PhLNJZ2; Figure 4 The results show the pH stability of bacteriophage PhLNJZ2. Figure 5 This represents the optimal multiplicity of infection (MLI) result for PhLNJZ2 bacteriophage. Figure 6 The results of the one-step growth curve determination of phage PhLNJZ2; Figure 7 This is a graph showing the distribution of reading segment lengths. Figure 8 This is a circumference diagram of the epigenetic modification distribution of phage PhLNJZ2; Figure 9 The image shows the electrophoresis results; in which: lane 1 is non-alginolytic Vibrio; lane 2 is alginolytic Vibrio (positive); lane 3 is the negative control; lanes 4-6 are other detected bacteria; lane 2 shows a specific amplification band at the expected position, while lanes 1 and 3-6 do not show this band. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] It should be noted that the terminology used herein is for describing specific embodiments only and is not intended to limit the exemplary embodiments of the present invention. To enable those skilled in the art to more clearly understand the technical solutions of the present invention, the technical solutions of the present invention will be described in detail below with reference to specific embodiments. In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the reagents, materials, and equipment used are all commercially available.

[0028] In this invention, the terms "multidrug-resistant Vibrio alginolyticus target bacteria" or "host bacteria" specifically refer to Vibrio alginolyticus strains that meet all of the following characteristics: Strain identification: 16S rRNA gene sequencing (primer sequences shown in SEQ ID NO.1 and SEQ ID NO.2) identified it as *Vibrio alginolyticus*. Vibrio alginolyticus The sequence similarity with the model strain is >99%.

[0029] Core multidrug resistance spectrum: Using the Kirby-Bauer disk diffusion method described in Example 1, resistance was observed to seven antibiotics: amoxicillin, amoxicillin-clavulanate potassium (4:1), clindamycin, neomycin sulfate, tylosin tartrate, cefixime, and fosfomycin calcium (R, inhibition zone diameter ≤10 mm). This resistance spectrum is a sufficient condition for screening the bacteriophage of this invention.

[0030] Source of isolation: It can be obtained from the hepatopancreatic tissue of aquatic animals (such as shrimp) exhibiting typical symptoms of vibriosis (such as empty intestine and stomach, and hepatopancreatic atrophy), according to the TCBS culture medium isolation and purification method disclosed in Example 1.

[0031] Example 1: Isolation, identification and drug resistance determination of pathogenic bacteria 1. Sample Source In May 2024, in a shrimp farming pond in Yancheng, Jiangsu Province, five sludge samples (200g each, taken from the four corners and the central area of ​​the pond to ensure coverage of different pollution distribution areas), five water samples (500mL each, corresponding to the sludge sampling area), and ten diseased shrimp were collected (individuals with empty intestines and stomachs and obvious symptoms of hepatopancreatic atrophy were selected, and hepatopancreatic tissue was taken for later use).

[0032] 2. Isolation and purification of pathogenic bacteria (1) Sample pretreatment: For sludge samples, add 1000 mL of sterile physiological saline at a volume ratio of 1:5, shake at 28℃ and 180 rpm for 30 min, and after thorough suspension, let stand for 1 h. Take the supernatant and filter it through a 0.22 μm filter membrane to remove impurities and non-target microorganisms. Collect the filtrate for later use. For aquaculture water samples, directly take the original water sample and filter it through a 0.22 μm filter membrane for sterilization. Collect the filtrate for later use. For hepatopancreas tissue, mix and grind the hepatopancreas tissue of 10 shrimp, add 5 mL of sterile physiological saline, centrifuge at 12000 rpm for 10 min, take the supernatant and filter it through a 0.22 μm filter membrane. Collect the filtrate for later use.

[0033] (2) Initial screening culture and isolation: 100 μL of the treatment solution of the above three samples were taken and inoculated into TCBS medium plates using the three-zone streak method. The plates were then inverted and incubated in a facultative anaerobic incubator (containing 5% CO2) at 37℃ for 14-16 h. Observation revealed that only the hepatopancreatic tissue samples of diseased shrimp showed dominant yellow, flat, smooth single colonies, which are consistent with the typical characteristics of Vibrio alginolyticus on TCBS medium.

[0034] (3) Purification culture: A single yellow colony was picked and inoculated into LBS liquid medium (containing 3% NaCl). After incubation at 37℃ and 180 rpm for 8-12 h, it was again inoculated into TCBS medium plates using the three-zone streak method. The plates were then inverted and incubated in a facultative anaerobic incubator (containing 5% CO2) at 37℃ for 14-16 h. The streak was repeated 3 times to obtain a pure culture with uniform morphology and neat edges. This pure culture was designated as the representative strain and referred to as strain Va30 in this paper for ease of description. It was then inoculated into LBS slant medium for storage. The strain grew well in LBS liquid medium, and the bacterial solution became uniformly turbid after 12 h of incubation at 37℃.

[0035] It should be noted that strain Va30 is only used to exemplify the implementation of the present invention, and any other Vibrio alginolyticus strain conforming to the definition of the present invention can equivalently replace strain Va30 for implementing the present invention. Those skilled in the art, based on the definitions and methods disclosed in this invention, can isolate Vibrio alginolyticus strains with the same core drug resistance spectrum from the environment.

[0036] 3. Identification of pathogenic bacteria (1) Morphological identification: A single colony of strain Va30 was stained with Gram and observed under a microscope. The strain was a Gram-negative short bacillus with a curved shape, which is consistent with the typical morphological characteristics of Vibrio alginolyticus.

[0037] (2) Molecular biological identification: Genomic DNA was extracted from strain Va30 and used as a template for PCR amplification using Vibrio alginolyticus specific primers (sequences: Va-F: 5-GTCACGGCAAAGTGCTTAGTG-3', as shown in SEQ ID NO.1; Va-R: 5'-TTGGAGCTGAGTGTTGTGGTC-3', as shown in SEQ ID NO.2). Electrophoresis revealed a specific band at approximately 668 bp in the amplified product, as shown in SEQ ID NO.2. Figure 9 The product was sequenced, and the obtained sequence was compared with GenBank database using BLASTn. The results showed that it was similar to Vibrio alginolyticus (…). Vibrio alginolyticus The sequence similarity of the type strain was higher than 99.5%. Based on morphological characteristics, strain Va30 was identified as Vibrio alginolyticus.

[0038] 4. Multidrug resistance assay (1) Preparation of bacterial culture: Strain Va30 was inoculated into LBS liquid medium and cultured at 37°C with shaking until the logarithmic phase was reached. The OD of the bacterial culture was then adjusted. 600 The value is 0.4~0.5 (corresponding to a colony count of 1~2×10⁻⁶). 8 CFU / mL, which is equivalent to 0.5 McFarland turbidity.

[0039] (2) Preparation of quality control strains: Escherichia coli (commercial strain available to the public) ATCC25922 was inoculated onto MH agar plates and cultured at 37°C for 16-24 hours. Single colonies were picked to prepare a bacterial suspension with a turbidity of 0.5 McFarland ratio. The suspension was then spread onto MH agar plates for antibiotic quality control to ensure the effectiveness of the culture medium and drug sensitivity test discs.

[0040] (3) Plate inoculation: Take 200 μL of strain Va30 bacterial suspension after adjusting the concentration, and evenly drop it onto MH agar plate according to the five-point distribution. Use a sterile cotton swab to densely streak in a "Z" pattern to spread it evenly. Rotate around the culture medium to ensure that there are no gaps at the edge. Wait for the bacterial suspension to dry completely.

[0041] (4) Antimicrobial susceptibility testing: Using sterile forceps, pick up 14 antimicrobial susceptibility test strips for aquatic-related antibiotics (including penicillins, cephalosporins, carbapenems, aminoglycosides, tetracyclines, quinolones, and sulfonamides), and evenly apply them to the surface of the plate. The center-to-center distance between adjacent strips should be no less than 24 mm, and the distance between the strips and the edge of the plate should be no less than 15 mm. Apply 3-4 strips to each plate, avoiding overlap. Set up 3 replicates for each group.

[0042] (5) Incubation and determination: Place the plates face up for 10-15 minutes to fix the paper discs, then invert them and incubate in a 37℃ constant temperature incubator for 16-24 hours. After incubation, place the plates on a black background and measure the diameter of the inhibition zone around each antibiotic susceptibility test strip using calipers. The criteria for determining the susceptibility of strains to each antibiotic are: extremely sensitive (S) ≥ 22 mm, highly sensitive (I) 16-21 mm, low sensitive 10-15 mm, and resistant (R) ≤ 10 mm. The test results are shown in Table 1.

[0043] Table 1. Results of drug susceptibility testing of strain Va30 to 14 antibiotics. Drug sensitivity test tablet number Drug Name Diameter of the inhibition zone (mm) Sensitivity determination 1# Amoxicillin 6.1 Drug resistance (R) 2# Amoxicillin-clavulanate potassium (4:1) 7.7 Drug resistance (R) 3# clindamycin 8.4 Drug resistance (R) 4# Neomycin sulfate 8.8 Drug resistance (R) 5# Apramycin sulfate 16.0 Gao Min (I) 6# Amikacin Sulfate 14.7 Low sensitivity 7# Cefepime hydrochloride 11.0 Low sensitivity 8# Cefotaxime sodium 17.2 Gao Min (I) 9# Tylenol Tartrate 9.2 Drug resistance (R) 10# Doxycycline hydrochloride 11.7 Low sensitivity 11# Cefixime 6.0 Drug resistance (R) 12# Ceftriaxone sodium 17.5 Gao Min (I) 13# Colistin sulfate 16.4 Gao Min (I) 14# Fosfomycin calcium 9.0 Drug resistance (R) Strain Va30 exhibited resistance to 7 out of 14 tested antibiotics, encompassing multiple categories including β-lactams, macrolides, and aminoglycosides, thus meeting the definition of a multidrug-resistant (MDR) strain. This strain has been used as a target host for subsequent screening of specific bacteriophages, providing a research foundation for addressing the challenge of controlling multidrug-resistant Vibrio alginolyticus in aquaculture.

[0044] Example 2: Isolation and purification of bacteriophage PhLNJZ2 1. Sample collection The target bacteria of multidrug-resistant Vibrio alginolyticus (e.g., the representative strain Va30 described in Example 1) that meet the definition of this invention are used as host bacteria.

[0045] 2. Sample pretreatment The process is carried out according to the method in Example 1.

[0046] 3. Enrichment culture Take 10 mL each of the pretreated filtrates from the above-mentioned sludge, water, and hepatopancreas tissue, and mix them separately with 10 mL of logarithmic phase bacterial culture (e.g., strain Va30). 600 Mix (0.4~0.5), add 80mL of LBS liquid medium, and incubate at 37℃ and 180rpm for 16h with shaking to allow the phage to proliferate in large quantities within the host bacteria; then centrifuge the culture at 8000rpm for 10min to remove the bacterial precipitate, and filter the supernatant again through a 0.22μm filter membrane to obtain a high-concentration phage enrichment solution.

[0047] 4. Purification culture (1) Serial dilution: The phage enrichment solution was diluted with sterile physiological saline for 10 minutes. -1 Up to 10 -6 Gradient dilution; (2) Plate preparation: Take 100 μL of each gradient dilution and mix it with 100 μL of host bacteria (e.g., strain Va30) in the logarithmic phase. Add 5 mL of melted 0.7% LBS upper agar (cooled to 50°C), mix quickly, and pour into a culture dish containing 1.5% LBS lower agar. Wait for the upper agar to solidify. (3) Cultivation and screening: Place the plate upside down in a 37℃ constant temperature incubator and incubate for 12 hours. Then, select single phage plaques with a diameter of 3-5 mm, regular edges, and high transparency. (4) Repeated purification: The picked single phage plaques were inoculated into 1 mL of LBS liquid medium and mixed with 500 μL of host bacterial culture for amplification. The serial dilution-plating-picking of single phage plaques was repeated 3 times until all phage plaques on the plate were of the same size, morphology and transparency, thus obtaining pure cultured phage PhLNJZ2. Figure 1 As shown.

[0048] The bacteriophage PhLNJZ2 was deposited on October 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20252251, and classified as follows: Vibrio phage PhLNJZ2.

[0049] It should be emphasized that the selection of strain Va30 as the representative host bacterium in this invention is solely for the convenience of experimental operation and the comparability of results. This invention is the first to discover that as long as the *Vibrio alginolyticus* strain possesses the core resistance spectrum of the seven antibiotics defined in Example 1 (resistant to amoxicillin, amoxicillin-clavulanate potassium, clindamycin, neomycin sulfate, tylosin tartrate, cefixime, and fosfomycin calcium), it can serve as an effective screening target, allowing for the replication of the phage isolation process of this invention. Those skilled in the art do not need to rely on strain Va30 itself; they only need to follow the resistance spectrum definition and isolation method disclosed in this invention to obtain functionally equivalent host bacteria from the environment and repeatedly obtain short-tailed phages with broad-spectrum cross-species lytic activity. Therefore, the feasibility of this invention does not depend on the preservation of strain Va30, but rather on the specific resistance spectrum-phage broad-spectrum correlation pattern disclosed for the first time in this invention.

[0050] Example 3: Morphological observation of bacteriophage PhLNJZ2 Take 20 μL of pure culture of PhLNJZ2 phage (titer ≥10). 8 Add (PFU / mL) drops to a copper grid, allow to stand at room temperature for 15 minutes to precipitate, and then blot away excess liquid with filter paper. Add 2% phosphotungstic acid staining solution and stain for 8 minutes, then blot away any residual staining solution and allow to air dry at room temperature.

[0051] The results were observed using a transmission electron microscope (80.0 kV, 80.0 kx). Figure 2 As shown (scale bar 200nm): Bacteriophage PhLNJZ2 has an icosahedral head, approximately 60nm in diameter, and a short, non-contractile tail, about 15nm long, connected to the head via a neck. According to the International Council for Taxonomy of Viruses (ICTV) classification, this bacteriophage is classified as belonging to the family Brachyphageidae in the order Twinovirales. Podoviridae ).

[0052] Example 4: Determination of the biological characteristics of bacteriophage PhLNJZ2 1. Temperature stability measurement The potency is 1.0 × 10 8 Phage suspension at PFU / mL PhLNJZ2 was aliquoted into 5 sterile EP tubes, 1 mL per tube, and incubated at 4℃, 25℃, 37℃, 50℃, and 60℃. Samples were taken at 0h, 6h, 12h, and 24h, and the titer at each time point was determined using the double-layer agar plate method. The activity retention rate was calculated (activity retention rate = post-treatment titer / initial titer × 100%), with 3 replicates per group.

[0053] The results are as follows Figure 3As shown, the activity retention rate of bacteriophage PhLNJZ2 was above 85% after incubation at 4-37℃ for 24 hours, and 28.5% at 50℃. After treatment at 60℃ for 6 hours, the retention rate dropped to 15.4%, and was below 0.5% after 12 hours. This indicates that the bacteriophage has good stability within the conventional temperature range of aquaculture and has the temperature tolerance basis for practical application.

[0054] 2. pH stability determination Adjust the pH of LBS medium to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0, respectively. After sterilization by filtration through a 0.22 μm filter membrane, dispense 900 μL into each tube. Add 100 μL of PhLNJZ2 phage suspension (final titer 1.0 × 10⁻⁶) to each tube. 7 After incubation at 37°C for 24 hours (PFU / mL), the potency was determined using the double-layer agar plate method, and the activity retention rate was calculated. Each group was set up with 3 replicates.

[0055] The results are as follows Figure 4 As shown, the activity retention rate of bacteriophage PhLNJZ2 was above 82% in the pH range of 5.0-9.0, with the highest retention rate (96.7%) at pH 7.0. The retention rate was approximately 42% at pH 4.0 and 10.0, and below 1% at pH 3.0 and 11.0. This indicates that the bacteriophage has good adaptability to the pH environment (6.5-9.0) of conventional aquaculture, providing crucial pH tolerance assurance for its practical application.

[0056] 3. Optimal Multiple of Infection (MOI) Measurement The prepared potency was 1.0 × 10⁻⁶. 9 Phage suspensions with PFU / mL and a concentration of 1.0 × 10⁻⁶ 7 Logarithmic-phase bacterial suspensions of host bacteria (e.g., strain Va30) at CFU / mL were prepared, with six MOIs (Multiple of Infections) at 0.001, 0.01, 0.1, 1, 10, and 100. 5 mL of LBS liquid medium was added to each gradient group, and the phage suspension and bacterial suspension were mixed in the corresponding proportions. The mixtures were incubated at 37°C with shaking at 180 rpm for 12 h, centrifuged at 5000 rpm for 10 min, and the supernatant was serially diluted. Phage titers were determined using the double-layer agar plate method, with three replicates per group. The MOI corresponding to the highest titer group was considered the optimal multiple of infection.

[0057] The results are as follows Figure 5 As shown, the phage titer reaches its peak at MOI=1, which is 2.8×10⁻⁶. 9 PFU / mL; at MOI=0.1 and MOI=10, the potency was 1.9×10⁻⁶. 9PFU / mL and 2.1×10 9 PFU / mL; the potency of the remaining MOI gradients was all below 1.5 × 10⁻⁶. 9 PFU / mL. The optimal multiplicity of infection for phage PhLNJZ2 was determined to be 1.

[0058] 4. One-step growth curve determination At the optimal MOI of 1, the phage suspension was mixed with the logarithmic phase culture of the host bacterium (e.g., strain Va30), incubated at 37°C for 15 min (adsorption phase), centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the precipitate was washed twice with sterile LBS medium to remove free phage. The precipitate was resuspended in 10 mL of fresh LBS medium, and cultured at 37°C with shaking at 180 rpm while timing was active. Samples were taken every 5 min from 0 to 30 min, and every 10 min from 31 to 90 min. After each sampling, the samples were immediately serially diluted, and the titer was determined using the double-layer agar plate method. A one-step growth curve was plotted, and the latency period, outbreak period, and outbreak amount (outbreak amount = stationary titer / initial host bacterium concentration) were calculated. The experiment was repeated 3 times.

[0059] The results are as follows Figure 6 As shown, the latency period of phage PhLNJZ2 is approximately 22 min, the outbreak period is 35 min (32-67 min), and the outbreak yield reaches 98 PFU / cell; after 67 min, it enters the plateau phase, and the titer remains at 1.2 × 10⁻⁶ during the stationary phase. 9 The concentration of PFU / mL indicates that the phage has high proliferation efficiency and can rapidly lyse the host bacteria.

[0060] Example 5: Genome sequencing and analysis of bacteriophage PhLNJZ2 Genomic DNA of phage PhLNJZ2 was extracted using the TIANamp viral DNA / RNA extraction kit, and the DNA purity was verified by electrophoresis to meet sequencing requirements.

[0061] Sequencing was performed using the PacBio Revio platform in CCS mode based on SMRT technology, achieving a sequencing accuracy of ≥99.9%. A total of 97,890 reads were obtained, with a total data volume of 1.56G and an N50 read length of 16,965bp. The data quality is excellent, and the read length distribution is as follows: Figure 7 As shown. Assembly was performed using Unicycler and Canu software respectively, yielding a single complete contiguous group with 100% genome integrity.

[0062] The genome is an uncircularized linear double-stranded DNA, 32,736 bp in size, with a GC content of 44.42%. Bioinformatics analysis predicted 37 open reading frames, indicating a compact genome structure. It contains a small number of repetitive sequences, but no ncRNAs (tRNA, rRNA, etc.) were predicted.

[0063] Using PacBio SMRT sequencing technology for direct detection of base modifications, m4C and m6A methylation modifications were detected, with the core motif being a GATC sequence. The distribution of epigenetic modifications is shown in the figure. Figure 8 As shown, the diagram illustrates the following: From the outside to the inside, each circle represents: genomic location; the distribution of positive strand modification sites using a 2000bp window and a 2000bp step size; the distribution of antisense strand modification sites using a 2000bp window and a 2000bp step size; GC content: Calculated using a 2000bp window and a 2000bp step size, the inward portion indicates that the GC content in that region is lower than the average GC content of the whole genome, and the outward portion is the opposite, with higher peak values ​​indicating a greater difference from the average GC content; GC preference: Calculated using a 2000bp window and a 2000bp step size, specifically using the algorithm GC / G+C, the inward portion indicates that the G content in that region is lower than the C content, and the outward portion is the opposite. Figure 8 This study visually demonstrates that methylation modification sites in the PhLNJZ2 genome are mainly concentrated in specific functional regions, and that the distribution of these modifications is correlated with GC content and GC preference. This epigenetic modification characteristic may contribute to enhancing the stability of phage DNA in complex culture environments and its ability to resist degradation by host endonucleases.

[0064] After comparison with the Virulence Factor Database (VFDB) and the Antibiotic Resistance Gene Database (CARD), no antibiotic resistance genes or virulence genes were found, indicating good safety.

[0065] BLASTn comparison showed that phage PhLNJZ2 and Vibrio Phage PVA1 (Genbank ID NC_023605.1) has a similarity of 95.51% and a coverage of 74%. Phylogenetic analysis shows that it is closely related to members of the family Brachyphageidae and is a new member of this family, thus it is identified as a new species.

[0066] Example 6: Host profile determination and lysis characteristics analysis of bacteriophage PhLNJZ2 1. Preparation of test strains Sixteen Vibrio strains that pose a serious threat in aquaculture were selected. Detailed information is shown in Table 2.

[0067] Table 2. Source and characteristics of the tested Vibrio strains strain number strain name source strain type Serotype / Genoid Va01 Vibrio alginolyticus Shrimp from Yancheng, Jiangsu Province (sick) Clinical isolates Genotype A Va02 Vibrio alginolyticus Sea cucumber farming ponds in Qingdao, Shandong Clinical isolates Genotype B Va03 Vibrio alginolyticus Silt from crab farming ponds in Ningbo, Zhejiang Clinical isolates Genotype A Va04 Vibrio alginolyticus Hainan Sanya shrimp hatchery seedlings Clinical isolates Genotype C Va30 (Representative strain of this invention) Vibrio alginolyticus Shrimp from Yancheng, Jiangsu Province (sick) Clinical isolates multidrug-resistant strains VaStd Vibrio alginolyticus China Microbial Culture Collection Center Standard strain ATCC 33787 Vp01 Vibrio parahaemolyticus Shrimp from Lianyungang, Jiangsu (red body disease) Clinical isolates O1 serotype Vp02 Vibrio parahaemolyticus Sea bass from Dongying, Shandong (enteritis) Clinical isolates O5 serotype Vp03 Vibrio parahaemolyticus Aquaculture water bodies in Zhoushan, Zhejiang Clinical isolates O3 serotype Vp04 Vibrio parahaemolyticus Oysters from Xiamen, Fujian Clinical isolates Unknown serotype VpStd Vibrio parahaemolyticus China Microbial Culture Collection Center Standard strain ATCC 17802 Vh01 Vibrio harveyi Sea cucumbers from Yantai, Shandong (with skin rot disease) Clinical isolates Genotype I Vh02 Vibrio harveyi Jiangsu Nantong prawns (swimming in ponds) Clinical isolates Genotype II Vh03 Vibrio harveyi Hainan Lingshui aquaculture silt Clinical isolates Genotype I Vh04 Vibrio harveyi Crabs from Taizhou, Zhejiang (with limb rot disease) Clinical isolates Genotype III VhStd Vibrio harveyi China Microbial Culture Collection Center Standard strain ATCC 33846 It should be understood that the test strains listed in Table 2 are only used to exemplify the lysis profile of bacteriophage PhLNJZ2 and are not intended to limit the scope of protection of this invention. Those skilled in the art can use Vibrio alginolyticus, Vibrio parahaemolyticus, or Vibrio harveyi strains from other sources with similar characteristics to verify the activity of this bacteriophage. The standard strains can be purchased from public collections, and clinical isolates can be obtained using the isolation and identification method disclosed in Example 1 of this invention.

[0068] All test strains were inoculated into LBS liquid medium (containing 3% NaCl) and cultured at 37°C with shaking at 180 rpm until the logarithmic growth phase (OD2). 600 =0.4~0.5, corresponding to a colony count of 1~2×10 8 (CFU / mL); using the representative strain Va30 of this invention as a positive control and sterile physiological saline as a negative control.

[0069] 2. Host profile determination method (drop method combined with double-layer agar plate method) (1) Culture medium preparation Bottom layer agar: Prepare LBS medium containing 1.5% agar, autoclave at 121℃ for 15 min, cool to 50℃ and pour 15 mL into each petri dish, let it solidify at room temperature for later use.

[0070] Upper agar: Prepare LBS medium containing 0.7% agar, sterilize and cool to 50°C for later use.

[0071] (2) Treatment of bacterial culture and bacteriophage Take 100 μL of the logarithmic phase bacterial culture of each test strain, add it to 5 mL of melted top agar and mix quickly. Immediately pour the mixture into the corresponding bottom agar culture dish, rotate it horizontally to ensure even coverage, and let it stand at room temperature for 30 min to air dry.

[0072] The potency is 1.0 × 10 9 Phage suspension at PFU / mL was serially diluted with sterile physiological saline to 1.0 × 10⁻⁶ PFU / mL. 6 PFU / mL, for later use.

[0073] (3) Gradually and Cultivation Using a sterile pipette, aspirate 5 μL of diluted phage suspension and evenly drop it onto the surface of the upper agar plate. Set up three replicates for each strain. For the positive control, drop the same concentration of phage suspension; for the negative control, drop 5 μL of sterile physiological saline. After the liquid has completely air-dried, invert the plate and place it in a 37°C incubator. Observe the results after 16–18 hours.

[0074] (4) Result judgment criteria Clear, transparent plaques with a diameter ≥3mm are classified as having "strong lytic activity (+++)"; Transparent phage plaques with a diameter of 1-3 mm are classified as having "moderate lytic activity (++)". The presence of a hazy halo and the absence of obvious transparent spots indicate "weak cleavage activity (+)"; The absence of any inhibition zone indicates "no lysis activity (-)".

[0075] 3. Measurement Results The positive control plates (strain Va30) all showed clear, transparent phage plaques with a diameter of 3-5 mm, while the negative control showed no inhibition zones. The lysis results of the 16 tested Vibrio strains are shown in Tables 3 and 4.

[0076] Table 3. Statistical analysis of the lysis effect of bacteriophage PhLNJZ2 on different types of Vibrio. strains Test strain Strongly lytic strain (strain) Medium-sized lysate strains (plants) Weakly lysed strain (strain) No lysed strains (strains) Total pyrolysis rate (%) Vibrio alginolyticus 6 4 1 0 1 83.3 Vibrio parahaemolyticus 5 3 1 0 1 80.0 Vibrio harveyi 5 2 1 1 1 80.0 total 16 9 3 1 3 81.25 Table 4. Lysis of typical Vibrio strains by bacteriophage PhLNJZ2 strain number strain name Diameter of the inhibition zone (mm) cleavage activity Key features Va30 (Representative strain of this invention) Vibrio alginolyticus 4.2 +++ multidrug-resistant strains Va01 Vibrio alginolyticus 3.8 +++ Clinical isolates from shrimp (genotype A) Vp01 Vibrio parahaemolyticus 3.5 +++ Red body disease strain in shrimp (O1 serotype) Vh01 Vibrio harveyi 3.2 +++ Sea cucumber skin rot disease strain (genotype I) Va04 Vibrio alginolyticus 2.5 ++ Seedling source from the nursery (genotype C) Vp03 Vibrio parahaemolyticus 2.2 ++ Water source (O3 serotype) Vh02 Vibrio harveyi 1.8 ++ Shrimp pond-dwelling disease strain (genotype II) 4. Results Analysis (1) Fragmentation spectral characteristics The phage PhLNJZ2 achieved a total lysis rate of 81.25% against 16 tested core pathogenic Vibrio strains in aquaculture, including 83.3% against Vibrio alginolyticus, 80.0% against Vibrio parahaemolyticus, and 80.0% against Vibrio harveyi. This phage showed no significant serotype / genotype bias, and its core lysis targets were all highly prevalent pathogenic strains in aquaculture, demonstrating its significant practical value.

[0077] (2) Application value analysis Vibrio diseases in aquaculture are often caused by mixed infections of multiple Vibrio species. For example, in shrimp farming, Vibrio alginolyticus and Vibrio parahaemolyticus cause synergistic pathogenicity, while in sea cucumber farming, Vibrio harveyi and Vibrio alginolyticus cross-infect. Traditional single-control methods are insufficient to address all these issues simultaneously. Phyplast PhLNJZ2 can simultaneously and efficiently cover three core pathogenic Vibrio species without the need for preparing a "phage cocktail" mixture, simplifying the application process and reducing the cost for small and medium-sized farms. Its high lysosomal ratio reaches 56.25% (9 / 16), medium lysomal ratio 18.75% (3 / 16), and low lysomal ratio 6.25% (1 / 16), indicating that most strains can be rapidly and efficiently lysed, meeting the needs of emergency disease control in aquaculture scenarios.

[0078] Example 7: Application of bacteriophage PhLNJZ2 in shrimp farming 1. Experimental Design Three standardized shrimp farming ponds with an effective water volume of 10 mu·m (area 10 mu, average water depth 1 meter) were selected and randomly divided into group A (blank control group), group B (conventional dosage group), and group C (high dosage group). Each group was stocked with 100,000 shrimp larvae with a body length of 2 cm. The farming environment was consistent (water temperature 25~28℃, salinity 20‰, dissolved oxygen ≥5mg / L, pH 7.8~8.6).

[0079] 2. Test treatment Group A was treated with an equal volume of sterile saline solution as the phage preparation dilution for Groups B and C; Group B was treated at a concentration of 0.8 g / m³. 3 The dosage of PhLNJZ2 bacteriophage preparation (potency ≥10) was evenly applied to the entire pond. 8 PFU / mL), administered continuously for 3 days; Group C was administered at 1.5 g / mL. 3 Administer the same dosage and method for 3 consecutive days.

[0080] 3. Detection indicators Water samples were collected before the experiment (day 0) and 1, 3, 7 and 14 days after the experiment. The concentration of multidrug-resistant Vibrio alginolyticus in the water was determined by the TCBS selective medium plate counting method. The survival rate and disease status (clinical symptoms such as empty intestine and stomach, swimming in the pond, and red body) of shrimp were recorded daily.

[0081] 4. Test Results The concentration of Vibrio alginolyticus in Group A water remained consistently at 10. 4 ~10 5 CFU / mL, shrimp survival rate was 65% during the test period; one day after administration of Group B, Vibrio concentration in the water decreased to 10. 3 When the concentration of Vibrio spp. was below CFU / mL and maintained at a low level for 14 days, the shrimp survival rate reached 88%. In Group C, the Vibrio spp. concentration in the water dropped to below 10 CFU / mL one day after application, and there was no significant rebound within 14 days. The shrimp survival rate was 92%, and there were no clinical symptoms of disease. The survival rates of Groups B and C were significantly higher than those of Group A (p<0.05).

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A broad-spectrum short-tailed bacteriophage PhLNJZ2 targeting multidrug-resistant Vibrio, characterized in that, The bacteriophage PhLNJZ2 was deposited on October 20, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan, China, with accession number CCTCC NO: M 20252251, and classified as follows: Vibrio phage PhLNJZ2.

2. The phage PhLNJZ2 according to claim 1, characterized in that, The genome of the phage PhLNJZ2 is a linear double-stranded DNA with a size of 32,736 bp, a GC content of 44.42%, and contains m4C and / or m6A methylation modifications.

3. The phage PhLNJZ2 according to claim 1 or 2, characterized in that, The bacteriophage PhLNJZ2 exhibits lytic activity against Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio harveyi.

4. A bacteriophage composition, characterized in that, It comprises the phage PhLNJZ2 as described in any one of claims 1 to 3, and a pharmaceutically, feed-oriented, or environmentally acceptable vector.

5. An aquatic feed additive, characterized in that, The bacteriophage PhLNJZ2 as described in any one of claims 1 to 3 or the bacteriophage composition as described in claim 4.

6. The use of PhLNJZ2 of any one of claims 1 to 3, the phage composition of claim 4, or the aquatic feed additive of claim 5 in the preparation of formulations for the prevention and / or treatment of vibriosis in aquatic animals.

7. The application according to claim 6, characterized in that, Vibrio infection is caused by multidrug-resistant Vibrio alginolyticus, Vibrio parahaemolyticus, and / or Vibrio harveyi.

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