A Vibrio harveyi bacteriophage and its application

Abstract

The present application discloses a Vibrio harveyi phage and its application; the present application isolates a new short-tailed phage Vibro PHZU0002 from seawater in the sea area around Daya Bay, Huizhou City, Guangdong Province, which has strong pathogenicity to Vibrio harveyi isolated from diseased grouper in Guangdong Marine Fisheries Experimental Center and has a tendency to be resistant to antibiotics such as tetracyclines, chloramphenicol, norfloxacin, etc., and has a wide lysis spectrum; and the phage is subjected to morphological, biological characteristics and whole genome analysis; the results show that the phage has a wide temperature adaptability range, and after the phage is treated within the range of room temperature to 50°C for 30 minutes, the phage still has a good lysis effect on bacteria, and at the same time has excellent pH stability; in addition, after the whole genome analysis is performed, the results show that the homology with the closest phage is only 38.53%; therefore, the phage Vibro PHZU0002 is a new phage.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to a Vibrio harveyi bacteriophage and its application. Background Art

[0002] The output of marine products in my country has been increasing year by year. However, the disease rate of marine aquaculture animals has increased, which seriously threatens the development of the global marine aquaculture industry. Vibrio harveyi is a Gram-negative bacterium with strong motility and virulence. It is widely distributed in the ocean and estuaries1. It can infect a variety of marine species such as Litopenaeus vannamei2, Pinctada martensii3, Paralichthys olivaceus4, etc. It can cause hemorrhagic sepsis, enteritis and eye lesions, and has a high mortality rate. It is one of the most frequently occurring pathogens in marine aquaculture animal disease incidents in recent years, and it seriously threatens the development of the global marine aquaculture industry5.

[0003] In the past, the use of antibiotics was an effective way to deal with pathogens such as Vibrio harveyi, but now pathogens have developed drug resistance, and the research and development of new antibiotics has entered a bottleneck period6. Therefore, in 2020, the Ministry of Agriculture and Rural Affairs and 10 other ministries and commissions jointly issued the "Several Opinions on Accelerating the Green Development of Aquaculture", which pointed out that the use of drugs in aquaculture should be reduced to delay the outbreak of drug-resistant bacteria. However, the current methods of replacing antibiotics for the treatment of farmed animals still have many defects such as adverse reactions, poor treatment effects, and unclear pharmacology. Therefore, finding new methods to safely and efficiently deal with pathogens in marine aquaculture and in-depth research on their treatment mechanisms are of great significance for marine aquaculture animals to deal with pathogenic bacteria infections and improve the marine economy.

[0004] In order to deal with the current serious problem of bacterial resistance and promote the development of aquaculture in accordance with the requirements of "improving quality and efficiency, and green development". A bacteriophage that was discovered a hundred years ago and can lyse bacteria before antibiotics has unexpectedly been re-emphasized by scholars7. Compared with antibiotics, the use of bacteriophages in marine aquaculture has the following advantages: 1) It is specific and will not affect other microorganisms in the environment; 2) It is easy to be biodegraded and is not easy to remain in the environment to make pathogens resistant; 3) Bacteriophages are ubiquitous in the aquatic environment, especially for marine Vibrio, there is a good phage isolation system, which is easy to separate and purify8,9. However, the narrow lysis spectrum of phages makes it difficult for them to be widely used like antibiotics. Therefore, we need to isolate more phages to fill the gaps in the narrow lysis spectrum.

[0005] References:

[0006] 1.Deng,Y.et al.Horizontal gene transfer contributes to virulence andantibiotic resistance of Vibrio harveyi 345basedon complete genome sequenceanalysis.BMC Genomics 20,761(2019).

[0007] 2.Liu,H.et al.Pathogen ofVibrio harveyiinfection and C-type lectinproteins in whiteleg shrimp(Litopenaeus vannamei).Fish ShellfishImmunol.119,554–562(2021).

[0008] 3.Lei,Q.N.et al.Molecular cloning and expression analysis of heatshock protein 20(HSP20)fromthe pearl oysterPinctadamartensii.Genet.Mol.Res.GMR 15,(2016).

[0009] 4.Yu,L.,Hu,Y.,Sun,B.&Sun,L.Immunological study ofthe outer membraneproteins of Vibrio harveyi:insights that link immunoprotectivity tointerference with bacterial infection.Fish ShellfishImmunol.35,1293–1300(2013).

[0010] 5. Kamble, MT et al. Depolymerized Fractions of Sulfated Galactans Extracted from Gracilaria fisheri and Their Antibacterial Activity against Vibrio parahaemolyticus and Vibrio harveyi. Mar. Drugs 20, 469 (2022).

[0011] 6. MacNair CR, Tsai CN, Rutherford ST, Tan MW, Returning to Nature for theNext Generation of Antimicrobial Therapeutics. Antibiotics 2023,12(8):1267.

[0012] 7. Strathdee SA, Hatfull GF, Mutalik VK, Schooley RT, Phage therapy: Frombiological mechanisms to future directions. Cell 2023,186(1):17–31.

[0013] 8.Luong T,SalabarriaAC,Edwards RA,Roach DR,Standardizedbacteriophagepurification for personalizedphage therapy.NatProtoc 2020,15(9):2867–2890.

[0014] 9.Kauffman KM, Chang WK, Brown JM, et al.Resolving the structure ofphage-bacteria interactions in the context of natural diversity.Nat Commun2022,13(1):372. Summary of the invention

[0015] The purpose of the present application is to provide a Vibrio harveyi phage and its application, which has good lysis property to Vibrio harveyi, greatly reduces the probability of Vibrio harveyi infection, and has a wide range of applications.

[0016] In a first aspect, the present application provides a Vibrio harveyi phage, which is named VibroPHZU0002 and is deposited in Guangdong Culture and Microbiological Collection Center with a deposit number of GDMCC NO: 65110-B1.

[0017] The phage was isolated from a diseased grouper at the Guangdong Marine Fisheries Experimental Center. It uses a strain of Vibrio harveyi as its host, which is highly pathogenic and resistant to antibiotics such as tetracyclines, chloramphenicol, and norfloxacin. A new short-tailed phage was isolated from the surrounding waters.

[0018] The double-layer agar plate culture method was further used to observe the infection of the phage to the host. After 16 hours, semi-transparent halo plaques of uniform size and about 0.8 mm in diameter were formed.

[0019] In a second aspect, the present application provides a lysis solution, which is a lysis solution comprising the Vibrio harveyi phage described above.

[0020] In a third aspect, the present application provides a phage preparation for inhibiting Vibrio harveyi, wherein the phage preparation comprises the active ingredient of the Vibrio harveyi phage Vibro PHZU0002 described above or comprises the lysate described above.

[0021] In a fourth aspect, the present application provides the above-mentioned Vibrio harveyi phage or lysis solution or phage preparation for inhibiting Vibrio harveyi, and use thereof in inhibiting Vibrio harveyi in non-disease diagnosis and treatment.

[0022] Preferably, the application is to prevent and control Vibrio harveyi in aquaculture diseases.

[0023] Preferably, the Vibrio harveyi phage or the lysate or the phage preparation is contacted with the Vibrio harveyi.

[0024] Preferably, the pH of the contact medium is 5-11.

[0025] Preferably, the treatment temperature during contacting is 20-50°C.

[0026] Preferably, the contact time between the contact medium and the Vibrio harveyi is at least 30 minutes.

[0027] Compared with the prior art, the beneficial effect of the present application is that the bacteriophage vibro PHZU0002 provided in the present application has been deposited in the Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: September 9, 2024, deposit number: GDMCC NO: 65110-B1, classification name Vibro PHZU0002. Transmission electron microscopy found that the bacteriophage is a short-tailed phage with a regular icosahedron head structure. Further testing of the phage showed that the phage has a wide range of temperature adaptability. After treating the phage for 30 minutes in the range of room temperature to 50°C, the phage still has a good lysis effect on bacteria. When the treatment temperature of the phage reaches 60°C, the lysis effect of the phage on bacteria is significantly reduced. In addition, the results show that treating the phage in the pH range of 5-11 does not affect the lysis effect of the phage on bacteria. When the pH reaches pH=3, the phage has no lysis effect on bacteria.

[0028] Biomaterial Deposit Information

[0029] The Vibrio harveyi phage Vibro PHZU0002 provided in this application is deposited in the Guangdong Provincial Microbiological Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit date: September 9, 2024, deposit number: GDMCC NO: 65110-B1. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0031] Figure 1 This is the plaque morphology of the bacteriophage Vibro PHZU0002 lysing Vibrio harveyi vhr-G2-2 in one of the embodiments of the present application;

[0032] Figure 2 This is a transmission electron microscope microscopic morphology observation picture of bacteriophage Vibro PHZU0002 in one embodiment of the present application;

[0033] Figure 3 This is a pH stability measurement chart of bacteriophage Vibro PHZU0002 in one embodiment of the present application;

[0034] Figure 4This is a temperature stability measurement chart of bacteriophage Vibro PHZU0002 according to one embodiment of the present application;

[0035] Figure 5 This is a diagram showing the optimal infection multiplicity determination of bacteriophage Vibro PHZU0002 according to one embodiment of the present application;

[0036] Figure 6 This is a one-step growth curve of bacteriophage Vibro PHZU0002 in one embodiment of the present application;

[0037] Figure 7 This is a genome composition diagram of bacteriophage Vibro PHZU0002 according to one embodiment of the present application. DETAILED DESCRIPTION

[0038] In order to make the purpose, advantages and features of the present invention more understandable, the specific implementation methods of the present invention are described in detail below in conjunction with embodiments. Several embodiments of the present invention are given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive:

[0039] The present application provides a Vibrio harveyi phage, which is named Vibro PHZU0002 and is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65110-B1.

[0040] This application uses a strain of Vibrio harveyi that is severely resistant to antibiotics such as tetracyclines, chloramphenicol, and norfloxacin as a host, and isolates a bacteriophage VibroPHZU0002 with a broad lysis spectrum against Vibrio harveyi from a marine water sample. The double-layer agar plate method was used to observe the infection of the phage to the host. After 16 hours, a semi-transparent halo plaque of uniform size and a diameter of 0.8 mm was formed. For details, see Figure 1 .

[0041] The present application further discovered through transmission electron microscopy that the phage is a short-tailed phage, with a head structure of a regular icosahedron, a diameter of about 60-70nm, and a tail length of about 20-30nm. For details, see Figure 2 .

[0042] The present application further studies the biological characteristics of the phage, and uses the double-layer agar plate spot method to determine the lysis spectrum of the phage plaque Vibro PHZU0002. The results show that the phage can infect 4 pathogenic bacteria isolated from the gills and liver of diseased grouper.

[0043] The present application further conducts stability tests on the isolated phage, including tests on thermal stability and pH stability. The results show that the phage has a wide range of adaptability to temperature. After the phage is treated at room temperature to 50°C for 30 minutes, the phage still has a good lysis effect on bacteria. When the treatment temperature of the phage reaches 60°C, the lysis effect of the phage on bacteria is significantly reduced. For details, please refer to Figure 3 At the same time, the results show that the phage treatment in the pH range of 5-11 does not affect the phage's lysis effect on bacteria. When the pH reaches pH=3, the phage has no lysis effect on bacteria. For details, please refer to Figure 4 .

[0044] See also Figure 6 The present application further studies the optimal infection multiplicity (MOI) of bacteriophage Vibro PHZU0002 infecting its host Vibrio harveyi, which is 10, and produces the highest progeny phage titer of 1.4x10 9 pfu / mL.

[0045] See also Figure 6 The present application used the optimal infection multiplicity (MOI=10) of the phage to explore the one-step growth curve of its infection host Vibrio harveyi. The results showed that the initial 20-minute incubation period of the phage infection host bacteria was the latent period, and after 70 minutes, it entered the lytic stable period.

[0046] Further information Figure 7 The genome of the phage was sequenced using the DNBSEQ-T7 platform. The sequencing results showed that the genome size of the phage was 47587 bp. For details, please refer to Seq_1 in the sequence table. The present application further analyzed the gene sequence and found that the phage was a short-tailed phage. No homologous phage was found through the BLAST comparison analysis of the whole genome sequencing. Therefore, the phage Vibro PHZU0002 is a new phage.

[0047] Based on the bacteriophage Vibro PHZU0002 isolated in the present application, the present application further provides a lysis solution, which contains the above-mentioned Vibrio harveyi bacteriophage Vibro PHZU0002.

[0048] Specifically, the present application further provides a phage preparation for inhibiting Vibrio harveyi, wherein the phage preparation comprises the active ingredient of the Vibrio harveyi phage Vibro PHZU0002 described above or comprises the lysate described above.

[0049] It should be noted that the present application has no particular limitation on the dosage form of the phage preparation, and any type of phage preparation known in the art may be used.

[0050] Furthermore, the phage preparation may further include excipients, and the excipients are routinely selected according to the dosage form of the preparation.

[0051] Specifically, it should be noted that the present application has no special restrictions on the preparation method of the phage preparation, and any preparation type known in the art can be used.

[0052] Specifically, the present application also provides a use of the above-mentioned Vibrio harveyi phage or lysate or phage preparation for inhibiting Vibrio harveyi in inhibiting Vibrio harveyi in non-disease diagnosis and treatment.

[0053] Furthermore, its specific application is to prevent and control Vibrio harveyi in aquaculture diseases.

[0054] Furthermore, the application method can be to contact the Vibrio harveyi phage or lysate or phage preparation with the Vibrio harveyi.

[0055] Furthermore, the pH of the contact medium is 4-11; and the pH of the contact medium is more preferably 5-11.

[0056] Furthermore, the treatment temperature during contact is 20-50°C.

[0057] Furthermore, the contact time between the contact medium and Vibrio harveyi is at least 30 minutes.

[0058] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0059] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0060] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0061] Example 1

[0062] Isolation, Purification and Observation of Bacteriophage Vibro PHZU0002

[0063] 1) Phage isolation

[0064] The present application separates a pathogenic bacterium from the gills of diseased grouper, and identifies it as Vibrio harveyi by sequencing its 16S rRNA gene, and names it as vhr-G2-2, and uses it as a phage host to isolate phage.

[0065] Next, the present applicant collected ocean water samples in Huizhou City, Guangdong Province, and collected the filtrate by centrifuging at 8000g for 10 minutes to obtain the supernatant, and then filtering the supernatant through a 0.22μm filter. 3ml of the filtrate was mixed with 3ml of double LB liquid culture medium containing 7% NaCl, and 200μl of overnight cultured host bacteria Vibrio harveyi vhr-G2-2 bacterial solution was added, and cultured at 28°C and 200rpm shaking overnight. Then, the culture solution was centrifuged at 8000g for 10 minutes, the supernatant was collected and filtered through a 0.22μm filter to collect the enriched phage suspension, and then 100μl of the collected phage suspension was incubated with 100μl of the host bacterial solution for 1 hour, and the double-layer plate method was used to obtain uniform-sized, semi-transparent halo plaques, and the plaques were dug out and dissolved in SM buffer (200mM NaCl 2 ,10mMMgSO 4 , 50 mM Tris-HCl, pH 7.5). The above steps were repeated four times to obtain a purified phage, which was named VibroPHZU0002.

[0066] 2) Phage morphological characteristics

[0067] The obtained bacteriophage VibroPHZU0002 suspension was dripped onto a copper grid coated with a carbon film, stained with a 2% potassium phosphotungstate solution, and the morphology of the bacteriophage was observed under a transmission electron microscope.

[0068] 3) Phage host spectrum determination

[0069] Six strains of Vibrio harveyi were isolated from the gills and liver of diseased grouper using Vibrio harveyi selective medium, and were named vhr-L1-1, vhr-L1-2, vhr-G2-2, vhr-L2, vhr-G4, and vhr-G3-1. 100 μl of these six strains of Vibrio harveyi were mixed with 4 ml of semi-solid LB medium and poured into a bacterial culture dish containing solid LB medium. After solidification, 10 μl of Vibro PHZU0002 phage liquid was added to the center of the medium, and the medium was incubated at 28°C overnight to observe whether there were plaques and determine the lysis spectrum of phage Vibro PHZU0002.

[0070] Example 2

[0071] Study on biological characteristics of Vibro PHZU0002

[0072] 1) Phage thermal stability assay

[0073] Treat bacteriophage VibroPHZU0002 at 26℃, 30℃, 40℃, and 50℃ for 1 hour, dilute the titer, drop the dilution onto a semi-solid LB plate containing 3.5% NaCl of Vibrio harveyi vhr-G2-2, and culture overnight at 28℃. Calculate the temperature-treated phage titer, phage titer = plaque number × dilution (PFU / ml), and test the temperature stability of the phage. For specific test results, see Figure 5 .

[0074] 2) Phage acid-base stability determination

[0075] The applicant treated the bacteriophage VibroPHZU0002 for 1 hour in different pH environments (pH=3, 5, 7, 9, 11), performed titer dilution, and dropped the dilution onto a semi-solid LB plate containing 3.5% NaCl containing Vibrio harveyi vhr-G2-2, and cultured overnight at 28°C. The temperature-treated phage titer was calculated, phage titer = plaque number × dilution (PFU / ml), and the acid-base stability of the phage was tested. The specific test results can be found in Figure 3 .

[0076] 3) Determination of the optimal multiplicity of infection (MOI) of phage

[0077] Mix the host bacterial solution in the logarithmic growth phase with the phage solution in a ratio of 0.001, 0.01, 0.1, 1, 10, then culture at 28°C, 200rpm for 4 hours, centrifuge the culture solution at 8000g for 10 minutes, take the supernatant and dilute it with SM buffer, drop the dilution on a semi-solid LB plate containing 3.5% NaCl of Vibrio harveyi vhr-G2-2, and culture it at 28°C overnight. Calculate the temperature-treated phage titer, phage titer = plaque number × dilution (PFU / ml), and detect the optimal infection multiplicity of the phage. For specific measurement results, see Figure 5 .

[0078] 4) One-step growth curve of bacteriophage

[0079] The host bacterial liquid in the logarithmic growth phase was mixed with the phage liquid at the optimal infection multiplicity (MOI=10), incubated at 28°C for 30 minutes, centrifuged the incubation liquid at 10000g for 5 minutes, and discarded the supernatant. The precipitate was resuspended in 4 ml of LB liquid culture medium, cultured at 28°C and 200 rpm with shaking, and samples were taken every 10 minutes from 0 minutes to 200 minutes, and the supernatant was centrifuged and titered. The dilution was dropped onto a semi-solid LB plate containing 3.5% NaCl containing Vibrio harveyi vhr-G2-2, and cultured overnight at 28°C. The temperature-treated phage titer was calculated, phage titer = plaque number × dilution (PFU / ml), and the one-step growth curve of the phage was detected.

[0080] Example 3

[0081] Extraction, purification and sequencing of bacteriophage Vibro PHZU0002 genome

[0082] The quality and concentration of the extracted and purified Vibro PHZU0002 genomic DNA were measured by Nanodrop and QSEP, and 1ug of the genomic DNA was cut and purified by enzyme digestion. The purified fragmented DNA was trimmed and dA was added, and then connected to the adapter, and then amplified by PCR to obtain a DNA library. After the library quality inspection was qualified, the DNBSEQ-T7 platform was used for PE150 sequencing, and the raw data obtained by sequencing will be used for subsequent bioinformatics analysis.

[0083] Use fastp to filter and quality control the obtained raw sequencing data, cut the adapter, remove low-quality reads and reads with a high N ratio, and obtain clean reads. Use metaSPAdes software to perform de novo assembly on the clean reads, and select different k-mer lengths for testing and obtain the best assembly results. Then use bwa software to align the clean reads to the assembled genome sequence for statistical coverage. Then use Prokka software to annotate the genome coding genes and tRNA, and use blastp to align the protein sequence with the NR library to obtain sequence information with high similarity for each gene in the NR library. Use eggNOG-mapper to perform functional annotations on genes, and blast to align the gene sequence with the VFDB database for homology. For specific results, see Figure 7 .

[0084] In summary, the Vibrio harveyi phage isolated in the present application has a wide range of adaptability to temperature. After the phage is treated in the range of room temperature to 50°C for 30 minutes, the phage still has a good lysis effect on bacteria. In addition, it has a strong tolerance to environmental alkalinity, and treating the phage in the pH range of 5-11 does not affect the lysis effect of the phage on bacteria. Furthermore, through gene sequencing analysis, the homology with the closest phage is only 38.53%. Therefore, the phage VibroPHZU0002 is a new phage.

[0085] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application may be combined with each other.

[0086] The above description is only a preferred embodiment of the present application and does not constitute any form of limitation to the present application. Any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present application are within the scope of the technical solution of the present application.

Claims

1. A Vibrio harveyi phage, characterized in that: The Vibrio harveyi bacteriophage is named VibroPHZU0002, and is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65110-B1.

2. A lysate, characterized in that: The invention relates to a lysate containing the Vibrio harveyi phage according to claim 1.

3. A bacteriophage preparation for inhibiting Vibrio harveyi, characterized in that: The phage preparation comprises the active ingredient of the Vibrio harveyi phage Vibro PHZU0002 according to claim 1 or comprises the lysate according to claim 2.

4. Use of the Vibrio harveyi phage according to claim 1, or the lysate according to claim 2, or the Vibrio harveyi phage preparation according to claim 3 for inhibiting Vibrio harveyi in non-disease diagnosis and treatment.

5. The use according to claim 4, characterized in that: The application is to prevent and control Vibrio harveyi in aquaculture diseases.

6. The use according to claim 4, characterized in that: The Vibrio harveyi phage or the lysate or the phage preparation is contacted with the Vibrio harveyi.

7. The use according to claim 6, characterized in that: The pH of the contact medium is 5-11.

8. The use according to claim 6, characterized in that: The treatment temperature during contact is 20-50°C.

9. The use according to claim 6, characterized in that: The contact time between the contact medium and the Vibrio harveyi is at least 30 minutes.

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