Application of bacteriophages and combined fungicides that infect Pseudoalteromonas and Vibrio shioyasaki

Abstract

The present invention belongs to the technical field of microbial control, and specifically relates to the application of a phage that cross-infects Pseudoalteromonas haloplanktis and Vibrio, and a combined bactericide. The phage is phage PSY-P that cross-infects Pseudoalteromonas haloplanktis and Vibrio; it has been deposited at the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, the deposit date is December 12, 2024, and the deposit number is: CCTCC NO: M 20242795. The effective titer of this phage is as high as 1×10<supgt;10< / supgt; PFU / mL, providing a new biological control means for the prevention and control of infectious diseases of Pseudoalteromonas haloplanktis and Vibrio in marine aquaculture animals, and having important application value and good practical application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial control, and specifically relates to a bacteriophage capable of cross-infecting Pseudoalteromonas shioyasaki and Vibrio and the application of a combined bactericide. Background Art

[0002] As a natural bacterial predator, bacteriophages reproduce quickly and are green and environmentally friendly. They show unique advantages and great application potential in solving the problem of multi-drug resistant bacteria. However, the host range of bacteriophages is narrow, and they can often only lyse pathogenic bacteria within the same genus, making it difficult to deal with common co-infection events of complex pathogens of different species. Therefore, we urgently need to obtain bacteriophages that can infect different pathogens across species and even across orders. Farmed animal diseases caused by infection with pathogenic bacteria of the genera Pseudoalteromonas and Vibrio have the characteristics of rapid onset, wide epidemic range, and strong pathogenicity, which cause huge economic losses to the aquaculture industry and seriously restrict the development of the aquaculture industry. Therefore, the isolation of bacteriophages that can infect Vibrio and Pseudoalteromonas bacteria across orders is of great significance for ensuring green and healthy marine aquaculture and the sustainable development of marine aquaculture under the background of "no antibiotics". However, there are no reports of bacteriophages that infect pathogenic bacteria of the genera Pseudoalteromonas and Vibrio across orders. Summary of the invention

[0003] In view of the above-mentioned deficiencies in the prior art, the present invention provides a bacteriophage PSY-P that cross-infects Pseudomonas shioyasaki and Vibrio and the use of a combined fungicide.

[0004] The present invention is achieved through the following technical solutions:

[0005] A bacteriophage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki has been deposited in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and is named as: Pseudoalteromonas shioyasakiensis phage PSY-P, the deposit date is December 12, 2024, and the deposit number is: CCTCC NO: M20242795.

[0006] The titer of the bacteriophage PSY-P that cross-infects Pseudoalteromonas and Vibrio shioyasaki is not less than 10 10 PFU / ml.

[0007] An application of the bacteriophage that cross-infects Pseudoalteromonas shioyasaki and Vibrio, and an application of the bacteriophage in the preparation of a fungicide for pathogenic bacteria.

[0008] The pathogenic bacteria are Pseudoalteromonas shioyasaki and / or Vibrio.

[0009] A bactericide for eliminating Pseudoalteromonas shioyasaki and Vibrio, the bactericide comprising the bacteriophage.

[0010] The fungicide is in the form of wettable powder, water dispersible granules or water suspension.

[0011] The fungicide also includes auxiliary materials acceptable in aquaculture.

[0012] The bactericide comprises one or more adjuvants acceptable in aquaculture selected from the group consisting of dispersants, stabilizers, fillers and solvents.

[0013] An application of the bacteriophage or the bactericide, and application of the bacteriophage or the bactericide in controlling Vibrio infection in aquaculture.

[0014] Beneficial effects of the present invention:

[0015] The present invention screened and obtained a virulent bacteriophage PSY-P that can infect a strain of Pseudomonas shioyasaki and multiple strains of Vibrio bacteria. The effective titer of the bacteriophage is as high as 1×10 10 PFU / mL, which provides a new biological control method for the prevention and control of infectious diseases of Pseudomonas shioyasaki and Vibrio spp. in marine aquaculture animals, and has important application value and good practical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a plate culture photograph of the bacteriophage PSY-P that cross-infects Pseudomonas shioyasaki and Vibrio in Example 2 of the present invention.

[0017] Figure 2 The bacteriophage PSY-P that infects Pseudoalteromonas shioyasaki and Vibrio in Example 3 of the present invention is respectively Pseudoalteromonas shioyasakiensis SE3(T) 、Vibrio alginolyticus NBRC 15630(T) and Vibrio scophthalmi Pyrolysis images of LMG 19158(T).

[0018] Figure 3 This is a transmission electron microscope photograph of the bacteriophage PSY-P that cross-infects Pseudomonas shioyasaki and Vibrio in Example 4 of the present invention.

[0019] Figure 4 The bacteriophage PSY-P that infects Pseudomonas shioyasaki and Vibrio in Example 6 of the present invention infects Pseudomonas shioyasaki Pseudoalteromonas shioyasakiensis 24 h effect curve of SE3(T).

[0020] Figure 5 The bacteriophage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki in Example 7 of the present invention infects VibrioVibrio scophthalmi 24 h effect curve of LMG 19158(T).

[0021] Figure 6 This is a comparison result of the average nucleotide consistency between the bacteriophage PSY-P that infects Pseudomonas and Vibrio shioyasaki in Example 8 of the present invention and the known database.

[0022] Figure 7 This is a diagram showing the effect of immersion treatment of Japanese shrimp with the bacteriophage PSY-P that cross-infects Pseudoalteromonas shioyasaki and Vibrio in Example 9 of the present invention.

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention. DETAILED DESCRIPTION

[0024] It should be noted that the following detailed descriptions are all illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. It should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention.

[0026] In view of this, in one embodiment of the present invention, a bacteriophage PSY-P that cross-infects Pseudomonas shioyasaki and Vibrio is provided.

[0027] Among them, the bacteriophage PSY-P that cross-infects a strain of Pseudomonas shioyasaki and multiple strains of Vibrio has been sent to the China Center for Type Culture Collection, Wuhan University, Wuhan, China. The preservation date is December 12, 2024, and the preservation number is: CCTCC NO: M 20242795.

[0028] The biological characteristics of the bacteriophage PSY-P that cross-infects Pseudomonas and Vibrio shioyasaki described in the present invention are as follows: under a transmission electron microscope, PSY-P appears to be composed of a regular hexahedral head with a diameter of about 50 ± 2 nm and a tail with a length of 136 ± 2 nm, and belongs to the family Myocaudal phage.

[0029] The Capsid sequence of the bacteriophage PSY-P that cross-infects Pseudomonas shioyasaki and Vibrio according to the present invention is shown in SEQ NO.1:

[0030]

[0031] According to the International Committee on Taxonomy of Viruses (ICTV)'s classification criteria for the identification of new bacteriophages, the bacteriophage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki is a new phage genus of Caudoviricetes.

[0032] The experiments show that the PSY-P of the present invention can lyse the host bacteria Pseudoalteromonas shioyasaki Pseudoalteromonas shioyasakiensis SE3(T) and Vibrio scophthalmi LMG 19158(T) 、 Vibrio alginolyticus NBRC 15630(T).

[0033] The titer of the bacteriophage PSY-P for cross-infecting Pseudoalteromonas and Vibrio shioyasaki is not less than 10 10 PFU / ml.

[0034] In another specific embodiment of the present invention, there is provided use of the bacteriophage PSY-P that cross-infects Pseudoalteromonas shioyasaki and Vibrio in the preparation of a bactericide for Pseudoalteromonas shioyasaki.

[0035] In another specific embodiment of the present invention, a bactericide for Pseudoalteromonas and Vibrio shioyasaki is provided, wherein the bactericide for Pseudoalteromonas and Vibrio shioyasaki comprises the above-mentioned bacteriophage PSY-P that cross-infects Pseudoalteromonas and Vibrio shioyasaki.

[0036] In another specific embodiment of the present invention, the formulation of the bactericide for Pseudoalteromonas shioyasaki and Vibrio is a wettable powder, a water dispersible granule or a water suspension.

[0037] In another specific embodiment of the present invention, the Shioyasaki Pseudoalteromonas and Vibrio fungicide further comprises an auxiliary material acceptable to the aquaculture industry, and the auxiliary material acceptable to the aquaculture industry is selected from one or more of a dispersant, a stabilizer, a filler and a solvent. There is no special restriction on the source of the auxiliary material acceptable to the aquaculture industry of the present invention, and generally commercially available products can be used.

[0038] In another specific embodiment of the present invention, there is provided the use of the above-mentioned cross-order infective Pseudoalteromonas and Vibrio phage of Shioyasaki or the Pseudoalteromonas and Vibrio bacteriocides of Shioyasaki in controlling Vibrio infection in aquaculture.

[0039] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0040] In the following examples, the formula of the 2216E medium is: 1 g yeast powder, 5 g peptone, 0.1 g ferric orthophosphate, 1000 mL Chenhai water, pH 7.6.

[0041] The 2216E solid culture medium comprises: 1 g yeast powder, 5 g peptone, 0.1 g ferric orthophosphate, 1000 mL Chenhai water, pH 7.6, and 15 g agar.

[0042] The 2216E semi-solid culture medium comprises: 1 g yeast powder, 5 g peptone, 0.1 g ferric orthophosphate, 1000 mL Chenhai water, pH 7.6, and 5 g agar.

[0043] Example 1 Isolation of bacteriophage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki

[0044] After filtering the nearshore seawater sample through a 0.22 μm filter membrane, the sample was added at a ratio of 50% to the host Pseudoalteromonas shioyasaki in the logarithmic growth phase. Pseudoalteromonas shioyasakiensis SE3(T) and Vibrio scophthalmi LMG 19158(T), Vibrio alginolyticus NBRC 15630(T), Vibrio parahaemolyticus NBRC12711(T) mixed bacterial solution, placed in a 28°C shaker for shaking culture. Monitor the OD of the mixed culture system every day. 600 Samples were taken and centrifuged at 1d, 2d, 3d, 4d, 5d, 6d, and 7d (8000 × g , 4°C, 10 min), discard the precipitate, collect the supernatant, filter through a 0.22 μm filter head, and dilute with SM buffer in different gradients. Take 1 mL of the logarithmic growth phase Pseudoalteromonas shioyasakiensis SE3(T) bacterial solution was mixed with 1 mL of filtrate with different dilution gradients, and adsorbed on a shaker at 28°C for 30 min. 5 mL of semi-solid culture medium melted and precooled to 48°C was added. After mixing, the mixture was poured onto a 2216E solid plate. After the upper agar solidified, the plate was placed in a 28°C incubator and cultured overnight. If plaques appeared, they were the phages that were initially isolated.

[0045] Example 2 Purification of bacteriophage PSY-P that infects Pseudoalteromonas shioyasaki and Vibrio

[0046] The plaque obtained in Example 1 was picked out and soaked in SM buffer, vortexed for 1 min to obtain a phage suspension, and the phage suspension was placed in a 4°C refrigerator and soaked overnight. The phage suspension was then filtered through a 0.22 μm filter head and graded diluted with SM buffer. Pseudoalteromonas shioyasakiensisSE3 (T) bacterial solution was used to make double-layer plates to obtain different plaques. This was repeated 3-5 times until the plaques on the plates were of the same size and shape. This is how purified phages were obtained. The morphology of the purified phage double-layer plates is shown in Figure 1. Figure 1 .

[0047] Example 3 Determination of the Lytic Host Range of the Cross-Order Infecting Pseudoalteromonas Shioyasaki and Vibrio phage PSY-P

[0048] Take 1 mL of each The bacterial suspensions in different logarithmic growth phases were mixed with 5 mL of semi-solid culture medium (specific bacteria are shown in Table 1), poured onto a solid plate, and 5 μL of the pure phage obtained in Example 2 was spotted on a double-layer plate, placed in a 28°C incubator for overnight culture, and observed for the appearance of plaques.

[0049] It has been verified that the phage PSY-P, which infects Pseudomonas shioyasaki and Vibrio spp., can not only lyse Pseudoalteromonas shioyasakiensis SE3(T), can also lyse Vibrio Vibrio scophthalmi LMG19158(T), against Vibrio Vibrio alginolyticus NBRC 15630(T) has a slight antibacterial effect, and the lysis morphology is shown in Figure 2 , cannot lyse Vibrio Vibrio parahaemolyticus NBRC 12711(T).

[0050] Table 1 Host range of bacteriophage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki

[0051]

[0052] Note: + means the test bacteria can be lysed by phage, - means the test bacteria cannot be lysed.

[0053] Example 4 Determination of the titer of PSY-P bacteriophage that crosses the order of infection with Pseudomonas shioyasaki and Vibrio

[0054] The pure phage obtained in Example 2 was used to prepare a double-layer plate according to the method of Example 1, and the plaques on the double-layer plate were counted. The plates with 30-300 plaques were selected to calculate the titer. The number of plaques on the plate was converted to the corresponding dilution factor, which was the titer of the phage. According to experimental determination, 100 μL diluted 10 8 The number of plaques formed by the phage solution was 146 on the double-layer plate. Therefore, the titer of the phage PSY-P that can cross-infect Pseudomonas shioyasakii and Vibrio was 1.46×10 10 PFU / mL.

[0055] Example 5 Electron microscopic morphological observation of PSY-P bacteriophage that crosses the order of infection with Pseudomonas shioyasaki and Vibrio

[0056] 1 mL of the pure phage liquid obtained in Example 2 was added to a 30 KD ultrafiltration tube for centrifugation, and the phage was washed 3 times with SM buffer. Then 2 μL of the pure phage sample was taken and dropped into the center of a 200-mesh copper mesh and fully dried in the air. After drying, it was stained with 2% uranyl acetate for 3 min, and then the phage morphology was observed using a Hitachi H-7800 transmission electron microscope with an accelerating voltage of 80 kV. The head width, tail length and width of the collected phage images were analyzed.

[0057] Electron microscopic morphology of bacteriophage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki Figure 3 The phage has a regular hexagonal head with a diameter of 50 ± 2 nm and a tail of about 136 ± 2 nm. From a morphological point of view, the phage PSY-P that infects Pseudomonas shioyasaki and Vibrio belongs to the family Myocaudaceae of the class Caudophaga.

[0058] Example 6 Cross-infection of Pseudomonas shioyasaki and Vibrio phage PSY-P Pseudoalteromonas shioyasakiensis SE3(T) bactericidal effect determination

[0059] According to the titer of bacteriophage PSY-P that infects Pseudomonas shioyasaki and Vibrio spp. measured in Example 4, a bacterial liquid of Pseudomonas shioyasaki in the logarithmic phase was Pseudoalteromonas shioyasakiensis SE3 (T) and phage suspension were placed in a shaker at 28°C with an MOI of 0.1. The control group was cultured with Pseudoalteromonas shioyasaki alone without any phage. Three parallels were set up. Starting from 0 h, samples were taken every 1 h to measure the OD of the bacterial solution. 600 The absorbance value was used to draw the growth curve of Pseudoalteromonas shioyasaki. Figure 4 The inhibition curve shows that compared with the control group, the experimental group had a significant antibacterial effect 3 hours after the addition of phages, which could last for 17 hours and reached the best antibacterial effect at 17.5 hours, with an inhibition rate of about 82.6%.

[0060] Example 7 Cross-infection of Pseudomonas shioyasaki and Vibrio phage PSY-P against Vibrio Vibrio scophthalmi Determination of the bactericidal effect of LMG 19158(T)

[0061] According to the titer of the bacteriophage PSY-P that infects Pseudomonas shioyasaki and Vibrio spp. determined in Example 4, the Vibrio spp. were isolated from the logarithmic phase using the method of Example 6. Vibrio scophthalmiThe growth curve of LMG 19158(T) is shown in Figure 5 . The obvious antibacterial effect began to appear 5 hours after the addition of bacteriophages, and the best antibacterial effect was achieved at 8 hours, with an inhibition rate of 59.5%.

[0062] Example 8 Genome Sequencing and Taxonomic Annotation of Phage PSY-P that Infects Pseudoalteromonas and Vibrio shioyasaki

[0063] The pure phage liquid in Example 2 was used to extract DNA using a viral genome kit (DNeasy Blood & Tissue Kit), and the operation method was performed according to the instructions of the kit. The DNA of the sample was sequenced using Illumina sequencing technology, and the effective reads were assembled using SPAdes software to obtain the genome of PSY-P, and the sequence is shown in SEQ NO.1. The gene sequence of PSY-P was compared with the Nt database, and after obtaining similar sequences, it was submitted to the VIRDIC online website to calculate the average nucleotide identity (ANI).

[0064] The whole genome sequence of phage PSY-P, which infects Pseudomonas and Vibrio shioyasaki, was compared with the GenBank database, and it was found that it had a high degree of identity (73.80%-85.76%) with the genomes of seven phage strains in the database. The average nucleic acid similarity of these eight phage genomes was further calculated, and the results were as follows: Figure 6 According to the International Committee on Taxonomy of Viruses (ICTV)'s criteria for the identification and classification of new phages (>75% considered to be the same genus), the phage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki has an ANI of <75% compared with the known phage genomes, and is a new phage genus of Caudoviricetes.

[0065] Example 9: Bath treatment of Japanese shrimp with the bacteriophage PSY-P that infects Pseudoalteromonas and Vibrio shioyasaki

[0066] Seawater was collected to culture Japanese shrimp in the laboratory for phage bath treatment experiments. The shrimp culture conditions were consistent with the average values ​​of seawater parameters: temperature was 20 ± 2℃, salinity was 3%, and pH was 7.6. After the seawater was aerated for 24 hours, four experimental groups were set up, with three parallels in each group and 20 healthy shrimp in each parallel. Group 1 was not added with any bacteria or preparations as the control group, and Group 2 was added with bacteria, including Pseudomonas shioyasaki and Vibrio, with concentrations of 10 6 CFU / mL), group 3 added bacteria and 10 7 Example 2 describes the cross-infection of Pseudomonas and Vibrio shioyasaki with the bacteriophage PSY-P suspension. Group 4 added bacteria and 10 6Example 2 of CFU / mL records the suspension of bacteriophage PSY-P that cross-infects Pseudoalteromonas and Vibrio shioyasaki, and observes the survival rate of Japanese shrimp after 5 days of cultivation (shrimp survival rate = number of survivals / total number of shrimps*100%). The amount and type of bacteria added in each group are the same.

[0067] After 5 days of immersion treatment, the survival rate of group 1 was 95±4.0%, the survival rate of group 2 was 6±2.3%, the survival rate of group 3 was 86±4.7%, and the survival rate of group 4 was 53±6.23%. The best treatment effect was MOI=10 ( Figure 7 ).

[0068] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, those skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A bacteriophage PSY-P that infects Pseudomonas shioyasaki and Vibrio across the order, characterized in that: The phage has been deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, with a deposit date of December 12, 2024, and a deposit number of CCTCC NO: M 20242795.

2. The bacteriophage that cross-infects Pseudoalteromonas shioyasaki and Vibrio according to claim 1, characterized in that: The titer of the bacteriophage PSY-P that cross-infects Pseudomonas shioyasaki and Vibrio is not less than 10 10 PFU / ml.

3. An application of the bacteriophage according to claim 1, characterized in that: The use of the bacteriophage in the preparation of a pathogenic bactericide, wherein the pathogenic bacterium is Pseudoalteromonas shioyasaki ( Pseudoalteromonas shioyasakiensis ), Vibrio maxima ( Vibrio scophthalmi ) and Vibrio alginolyticus ( Vibrio alginolyticus ) one or more of them.

4. A bactericide for removing Pseudoalteromonas shioyasaki and Vibrio, characterized in that: The bactericide comprises the bacteriophage according to claim 1.

5. The bactericide according to claim 4, characterized in that The fungicide is in the form of wettable powder, water dispersible granules or water suspension.

6. The bactericide according to claim 4, characterized in that The fungicide also includes auxiliary materials acceptable in aquaculture.

7. The bactericide according to claim 6, characterized in that The bactericide comprises one or more auxiliary materials acceptable in aquaculture selected from fillers and solvents.

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