Vibrio parahaemolyticus phage and application thereof

Abstract

The application discloses a Vibrio parahaemolyticus bacteriophage and application thereof, and the classification and name of the bacteriophage are VP01. Vibrio phage VP01, which is preserved in the China Center for Type Culture Collection on July 14, 2025, and has a preservation number of CCTCC M 20251601. The bacteriophage is a novel bacteriophage, encodes 62 ORFs, has no gene related to virulence function, and has high application safety; the bacteriophage can infect Vibrio parahaemolyticus to form clear bacterial plaques; the bacteriophage has strong lytic activity on Vibrio parahaemolyticus; and the bacteriophage is easy to preserve and can stably exist in a neutral environment at about 37 DEG C.

Description

Technical Field

[0001] This invention relates to the field of biological control, specifically to a Vibrio parahaemolyticus bacteriophage and its applications. Background Technology

[0002] Vibrio parahaemolyticus is a Gram-negative halophilic bacterium widely distributed in marine environments and estuaries. It is one of the leading causes of food poisoning globally, posing a serious threat to human health. In aquaculture, with the continuous expansion of farming scale and increasing stocking density, outbreaks of vibrio infection are becoming increasingly frequent, causing significant economic losses to the aquaculture industry. Vibrio infection not only has a high morbidity rate but also often leads to the death of large numbers of farmed organisms, severely impacting the sustainable development of aquaculture.

[0003] Traditional antibiotic control is prone to drug resistance and residues that endanger food safety, while biological control has attracted much attention from experts and scholars due to its advantages such as being green and environmentally friendly, having good therapeutic effects, and being economical. Among them, phage therapy, with its high specificity, rapid effect, non-toxicity, and pollution-free characteristics, has received special attention.

[0004] Bacteriophages are a type of virus that infects prokaryotes. They can specifically lyse host bacteria, reducing the number of host bacteria in water. This characteristic means that bacteriophages do not affect other microorganisms in the surrounding environment and have no adverse impact on the microecological environment. However, during the application of bacteriophages, insufficient lytic activity can lead to bacteria gradually developing resistance. Furthermore, some bacteriophages carry virulence genes or drug resistance genes, posing a risk of horizontal transmission. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a Vibrio parahaemolyticus phage and its application. This Vibrio parahaemolyticus exhibits strong lytic activity and high lytic yield, high tolerance to pH and temperature, and its genome lacks virulence-related genes, thus ensuring high safety.

[0006] An embodiment of the present invention proposes a Vibrio parahaemolyticus bacteriophage, the bacteriophage being classified and named as follows: Vibrio phage VP01 was deposited at the China Center for Type Culture Collection on July 14, 2025, with accession number CCTCC M20251601.

[0007] According to an embodiment of the present invention, a novel Vibrio parahaemolyticus phage is disclosed. This phage encodes 62 ORFs, has no genes related to virulence function, and exhibits high safety in application. The phage can infect Vibrio parahaemolyticus to form clear plaques and possesses strong infectivity against the bacteria. It also exhibits strong lytic activity against Vibrio parahaemolyticus, with a lysis rate reaching 200 PFU / cell. Furthermore, the phage is easily preserved and remains stable in a neutral environment at approximately 37°C.

[0008] Optionally, the bacteriophage can maintain stable activity at pH 5-9 and temperatures of 40℃-60℃.

[0009] In a second aspect, the present invention provides a phage preparation comprising the above-described Vibrio parahaemolyticus phage.

[0010] In a third aspect, this invention provides a drug for antagonizing Vibrio parahaemolyticus, comprising the aforementioned Vibrio parahaemolyticus phage and a pharmaceutically acceptable carrier. The Vibrio parahaemolyticus phage can inhibit the population growth of Vibrio parahaemolyticus, therefore the drug can be used to antagonize Vibrio parahaemolyticus. The drug can be produced according to methods well known to those skilled in the art and / or is commercially available. The drug can be in the form of a solvent, dispersant, suspension, granules, etc. It can be applied by spraying or other methods.

[0011] In a fourth aspect, this invention proposes a phage cocktail comprising the aforementioned Vibrio parahaemolyticus phage. The phage cocktail may include one or more Vibrio parahaemolyticus phages different from the aforementioned Vibrio parahaemolyticus phages; the combined use of different phages, in a cocktail-like manner, expands the host spectrum of the phages and improves the control effect against Vibrio parahaemolyticus.

[0012] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0013] Figure 1 A phage photograph of phage Xw-2 according to an embodiment of the present invention; Figure 2 A transmission electron microscope image of bacteriophage Xw-2 according to an embodiment of the present invention; Figure 3 This is a one-step growth curve of bacteriophage Xw-2 according to an embodiment of the present invention; Figure 4 This is a stability graph of bacteriophage Xw-2 under different pH conditions according to an embodiment of the present invention; Figure 5 This is a stability graph of bacteriophage Xw-2 under different temperature conditions according to an embodiment of the present invention; Figure 6 This is a diagram illustrating the inhibitory effect of bacteriophage Xw-2 on Vibrio parahaemolyticus according to an embodiment of the present invention. Figure 7 This is a genome circle diagram of bacteriophage Xw-2 according to an embodiment of the present invention. Detailed Implementation

[0014] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0015] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0016] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0017] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0018] Example 1: Isolation and Preparation of Bacteriophages Isolation and identification of Vibrio parahaemolyticus: (1) Select Litopenaeus vannamei ponds during the outbreak of vibrio disease (culture environment: salinity 20-30‰, water temperature 25-30℃, pH 7.5-8.5), collect silt from the bottom of the pond at 5-10cm (sample size ≥5g), place the samples in sterile centrifuge tubes, and transport them at 4℃ (process within 24 hours).

[0019] (2) Add the collected sludge and physiological saline (formula: 0.9% NaCl) at a ratio of 1g / 5mL into a 50mL centrifuge tube, shake thoroughly, and obtain the bacterial solution.

[0020] (3) Take the bacterial solution and perform a 10-fold serial dilution (10 -1 Up to 10 -6After being spread on TCBS plates and incubated at 37°C for 24 h, ten bright green single colonies from different plates were picked, purified five times by streaking on fresh TCBS plates, and DNA was extracted. After amplification of 16S rRNA, the samples were sent to Shanghai Sangon Biotech for identification. Molecular identification and biochemical characterization confirmed them to be Vibrio parahaemolyticus, and they were named Vibrio parahaemolyticus V1-V10.

[0021] It should be noted that V1-V10 used in this embodiment are Vibrio parahaemolyticus strains with taxonomic and key functional characteristics isolated by the above method. Those skilled in the art can follow the above steps to isolate strains that meet the characteristics from any shrimp pond with an outbreak of vibrio disease and use them to replace V1-V10 in this application to implement the present invention, and the experimental results are consistent.

[0022] Using Vibrio parahaemolyticus V8 as an indicator strain, bacteriophages were isolated from the bottom sludge of diseased shrimp ponds in aquaculture farms.

[0023] Collected sludge and SM buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 8 mM MgSO4·7H2O) were added to 50 mL centrifuge tubes at a ratio of 1 g / 5 mL. After thorough shaking, the tubes were placed in a 4°C refrigerator overnight. The next day, the tubes were centrifuged and filtered to collect the primary phage filtrate. This filtrate was then mixed with 2×2216E liquid medium at a volume ratio of 1:1 in a 50 mL centrifuge tube, and finally, 1% of Vibrio parahaemolyticus V8 was added. The tubes were incubated at 37°C and 180 rpm for 6 h on a shaker. After centrifugation at 12000 rpm for 10 min, the supernatant was collected and filtered through a 0.22 μm sterile filter membrane. 3 μL of the filtrate was spotted onto a double-layer plate (the upper layer being a mixture of semi-solid 2216E medium and Vibrio parahaemolyticus V8 bacterial suspension at a ratio of 100:1). The double-layer plates were incubated at 37°C for at least 12 h until transparent patches appeared on the plates. Figure 1 As shown.

[0024] Collect the plaque-forming filtrate and obtain individual plaques through a serial dilution method (ten-fold serial dilution). Pick the plaques and incubate them in fresh Vibrio parahaemolyticus V8 bacterial culture for 6 h. Centrifuge at 12000 rpm for 10 min, collect the supernatant, and filter it through a 0.22 μm sterile filter membrane to obtain a phage suspension. Repeat this step (serial dilution to obtain plaques) three times to obtain a pure culture of phage. Figure 2 The image shows the purified phage, denoted as Xw-2.

[0025] like Figure 2As shown, bacteriophage Xw-2 has an icosahedral head and a flexible tail, making it a typical tailed bacteriophage. This bacteriophage is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M 20251601; address: Wuhan University, Wuhan, China; deposit date: July 14, 2025; and classified as follows: Vibrio phage VP01.

[0026] Example 2: Host spectrum identification of bacteriophage Xw-2 Ten Vibrio parahaemolyticus strains (see Table 1, including V8 and other Vibrio parahaemolyticus strains isolated in the laboratory) were selected as indicator bacteria, and the host spectrum of bacteriophage Xw-2 was identified by double-layer plate spot method.

[0027] Take 1 mL of the cultured Vibrio parahaemolyticus V8 bacterial suspension and mix it with 5 mL of semi-solid 2216E medium. Pour the mixture into a Petri dish already coated with solid NB medium. After the upper layer cools, take 5 μL of the prepared bacteriophage Xw-2 suspension (obtained in Example 1) and spot it in the center of the Petri dish. Place the Petri dish in a 37℃ incubator and incubate for 12-24 h. If a plaque forms at the spotted location of the bacteriophage suspension, it indicates that the bacteriophage can infect the strain. The results are shown in Table 1.

[0028] The Xw-2 bacteriophage strain showed no lytic activity against several other bacteria, indicating that it is highly specific and relatively safe.

[0029] Table 1. Host spectrum of bacteriophage Xw-2.

[0030]

[0031] Note: + indicates that obvious phagocytic plaques can be formed; - indicates that obvious phagocytic plaques cannot be formed. Example 3: One-step growth curve of bacteriophage Xw-2 Take an appropriate amount of bacteriophage Xw-2 suspension (approximately 4 × 10⁻⁶). 7 PFU was added to 100 mL of Vibrio parahaemolyticus V8 bacterial culture in the logarithmic growth phase (approximately 4 × 10⁻⁶ PFU). 8The multiplicity of infection (MOI) was set to 0.1 (CFU). The mixture of phage and bacterial culture was allowed to stand for 30 min to allow for complete adsorption of the phage onto the cells. The cells were then centrifuged at 12000 g for 1 min, and the supernatant (containing unadsorbed phage, for which its titer was calculated; the number of infected bacterial cells was the initial total amount of phage added minus the total amount of phage remaining in the supernatant) was collected. The adsorbed bacterial cells were resuspended in 100 mL of fresh 2216E medium and cultured on a shaker at 33°C and 220 rpm. Then, 2 mL of bacterial culture was collected every 10 minutes, and after 60 minutes, 2 mL was collected every 20 minutes until 2 hours. The culture was then centrifuged at 12000 g for 1 min, and the phage titer in the supernatant was determined using the double-layer plate method until the phage titer stabilized. A one-step growth curve was plotted based on the phage titer at each time point.

[0032] The results are as follows Figure 3 As shown, the one-step growth curve indicates that after bacteriophage Xw-2 infects bacteria, the incubation period is approximately 20 minutes, followed by an exponential growth phase, reaching the lytic stationary phase 100 minutes after infection, with a final concentration of 4 × 10⁻⁶. 10 The phage lysis rate was 200 PFU / cell, indicating that phage Xw-2 has strong lytic activity against host bacteria.

[0033] Example 4: pH stability of bacteriophage Xw-2 Take 100 μL of the phage Xw-2 suspension prepared in Example 1 and add it to 900 μL of SM buffer solution with pH values ​​of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13. Mix well and place in a constant temperature water bath at 37°C for 1 h. Then, measure the titer under each pH condition.

[0034] The results are as follows Figure 4 As shown, the titer of bacteriophage Xw-2 remained almost unchanged under pH conditions of 5-9, indicating that bacteriophage Xw-2 can exist stably under natural environmental conditions.

[0035] Example 5: Temperature stability of bacteriophage Xw-2 Take the fresh phage Xw-2 suspension obtained in Example 1 and determine its initial titer. Then divide the phage Xw-2 suspension into 5 tubes and incubate them in water baths at 40℃, 50℃, 60℃, 70℃ and 80℃ respectively. After 1 hour, take the incubated phage suspension for titer determination.

[0036] The results are as follows Figure 5As shown, the titer of bacteriophage Xw-2 did not change significantly at temperatures of 40℃, 50℃, and 60℃. However, the titer dropped sharply at 70℃ and 80℃. Therefore, bacteriophage Xw-2 can exist stably under natural environmental conditions.

[0037] Example 6: Inhibitory effect of bacteriophage Xw-2 on Vibrio parahaemolyticus Take the Xw-2 suspension obtained in Example 1 and 100 μL of logarithmic growth phase (OD) 600 Add approximately 0.5-0.8 g of Vibrio parahaemolyticus V8 bacterial suspension (MOI approximately 0.1) to 5 mL, incubate at 33°C and 180 rpm on a shaker, and then measure the OD of the bacterial suspension every hour. 600 It lasted for 9 hours.

[0038] The results are as follows Figure 6 As shown, after adding bacteriophage Xw-2 to Vibrio parahaemolyticus V8 bacterial suspension, the OD value of the suspension remained unchanged until 5 hours later, while the OD value of the control group without bacteriophage addition continued to increase. This indicates that bacteriophage Xw-2 can reduce the host bacterial population to an extremely low level and has a strong ability to lyse the host bacteria, making it of great application value in the biocontrol of Vibrio parahaemolyticus infection.

[0039] Example 7: Extraction, whole-genome sequencing, and analysis of the Xw-2 phage genome. To further explore the characteristics of bacteriophage Xw-2, understand its related functional genes and whether it contains any risk genes, the genetic background of Xw-2 was further analyzed.

[0040] This embodiment uses the phenol-chloroform method to extract bacteriophage DNA: (1) Take 600 μL of high-titer phage preparation solution (greater than 1×10⁻⁶). 10 Add 3 μL of DNase and RNase A to a 1.5 mL centrifuge tube (PFU / mL), then add 3 μL of DNase and RNase A to a final concentration of 1 mg / mL. Mix well and incubate at 37°C for 1 h to degrade residual host bacterial DNA and RNA.

[0041] (2) Add 24 μL of 0.5% EDTA (pH 8.0), mix well, and inactivate at 80℃ for 15 min to inactivate the nuclease.

[0042] (3) Add 1.5 μL of 20 mg / mL proteinase K and 30 μL of 10% SDS, mix well, and incubate in a water bath at 56 °C for 1 h to digest the protein and lyse the phage.

[0043] (4) Add an equal volume (650 μL) of Tris-balanced phenol (pH 8.0), mix well, extract nucleic acid, centrifuge at 12000 r / min for 10 min, and transfer the upper aqueous phase to a new centrifuge tube.

[0044] (5) Add an equal volume of DNA / RNA extraction buffer (phenol: chloroform: isopropanol = 25:24:1), mix well, extract nucleic acid, centrifuge at 12000 r / min for 10 min, and transfer the upper aqueous phase to a new centrifuge tube.

[0045] (6) Add an equal volume of chloroform, mix well, remove residual phenol, centrifuge at 12000 r / min for 10 min, and transfer the upper aqueous phase to a new centrifuge tube.

[0046] (7) Add an equal volume of isopropanol, mix well, place at -20℃ for 3 h to precipitate nucleic acid, centrifuge at 13000 r / min for 20 min, slowly discard the supernatant and collect the precipitate.

[0047] (8) Add 1 mL of pre-cooled 75% ethanol, let stand for 10 min to precipitate nucleic acid, centrifuge at 12,000 r / min for 10 min, slowly pour off the ethanol and collect the precipitate.

[0048] (9) Dry at room temperature for 10 min to evaporate ethanol, then dissolve the nucleic acid precipitate in nuclease-free double-distilled water. Detect the genomic DNA concentration using an ELISA reader, and then measure the OD... 260 / OD 280 The pH value was controlled between 1.8 and 2.0. The nucleic acid quality was verified by gel electrophoresis, and finally the genome was sent to a sequencing company for testing.

[0049] Phage whole-genome sequencing employed a whole-genome shotgun (WGS) strategy to construct libraries with different insert fragments. Next-generation sequencing (NGS) was used on the Illumina NovaSeq platform to perform paired-end (PE) sequencing on these libraries, with read lengths of 150 bp. The raw sequencing data was processed to remove adapter contamination, filtering out reads shorter than 50 bp, reads with an average quality below 20, and reads with an n-to-number greater than 3, obtaining reads suitable for assembly. The phage genome was then assembled using ABySS software. High-frequency sequences in the high-throughput sequencing data were analyzed to determine the phage terminal sequences, ultimately obtaining the complete phage genome sequence. The entire genome was annotated using RAST (https: / / rast.nmpdr.org / ), and conserved domains of the coding sequences of each gene were predicted and analyzed.

[0050] like Figure 7 As shown, bacteriophage Xw-2 is a dsDNA genome with a full length of 39846 bp and a GC content of 42.91%. Sequence alignment was performed using the blastn program on the NCBI website. The results showed that bacteriophage Xw-2 had the highest similarity to Vibriophage P23, with an alignment coverage of 46% and a consistency of 91.35%. Bacteriophage Xw-2 is a novel bacteriophage.

[0051] Genome prediction analysis revealed that phage Xw-2 encodes 62 ORFs, while annotation and conserved domain analysis showed that only 22 ORFs have potential functions. Related functional protein genes are also clustered, as shown in Table 2. This phage contains genes related to lysis and is a virulent phage, which is advantageous in phage applications.

[0052] Whole-genome annotation analysis showed that there were no virulence-related genes in the genome of bacteriophage Xw-2, which provides a safety basis for the application of this bacteriophage.

[0053] Table 2. ORF annotation analysis of the Xw-2 phage genome.

[0054] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0055] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A Vibrio parahaemolyticus bacteriophage, characterized in that, The classification and naming of bacteriophages Vibrio phage VP01 was deposited at the China Center for Type Culture Collection on July 14, 2025, with accession number CCTCC M 20251601.

2. The Vibrio parahaemolyticus phage as described in claim 1, characterized in that, The bacteriophages maintained stable activity at pH 5-9 and temperatures 40℃-60℃.

3. A phage preparation, characterized in that, Includes the Vibrio parahaemolyticus phage as described in claim 1 or 2.

4. A drug antagonizing Vibrio parahaemolyticus, characterized in that, Includes the Vibrio parahaemolyticus phage as described in claim 1 or 2 and a pharmaceutically acceptable carrier.

5. A phage cocktail, characterized in that, Includes the Vibrio parahaemolyticus phage as described in claim 1 or 2.

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