CN120866235A - Novel virulent vibrio parahaemolyticus phage vB_ VpS _CC6 and application thereof - Google Patents

Abstract

The invention belongs to the technical field of microbial antibacterial preparations, and particularly discloses a novel vibrio parahaemolyticus phage isolate vB_ VpS _CC6 and application thereof. The phage vB_ VpS _CC6 was deposited at the cantonese microorganism strain collection at 9/14/2020 under the accession number GDMCC NO:61189-B1. The phage has good tolerance to temperature, pH, ultraviolet and other environments, and has good prevention and control effects on aquatic product pollution caused by vibrio parahaemolyticus. The phage has a lysis effect on multi-drug-resistant vibrio parahaemolyticus, can replace antibiotics to play a role in bacteriostasis, effectively solves the drug resistance problem faced by the current antibiotic treatment, and has good utilization value and application prospect. The phage can also be prepared into a composition with other phage to play a synergistic effect, and can be used for a broader-spectrum antibacterial or bacteriostatic application.

Description

Novel virulent vibrio parahaemolyticus phage vB_ VpS _CC6 and application thereof

Technical Field

The invention belongs to the technical field of microbial antibacterial preparations, and particularly relates to a novel vibrio parahaemolyticus phage vB_ VpS _CC6 and application thereof.

Background

Vibrio parahaemolyticus (Vibrio parahaemolyticus, VP) is a halophilic gram-negative bacterium and is widely distributed in marine products such as oceans, estuaries, and fish, shrimp and shellfish. Vibrio parahaemolyticus is a common food-borne pathogenic bacterium, and food poisoning is easily caused if food polluted by the pathogenic bacterium is eaten, so that digestive tract symptoms such as diarrhea, abdominal pain, nausea and vomiting are caused. Vibrio parahaemolyticus is capable of producing thermolabile haemolyticus TLH, thermostable direct haemolyticus TDH and thermostable direct haemolyticus-related toxin TRH, and type III secretion systems 1 (T3 SS 1) and 2 (T3 SS 2). TDH can exhibit biological activities such as cytotoxicity, hemolytic activity, cardiotoxicity, and lethality, whereas TRH has hemolytic and enterotoxic effects, and TRH is similar to TDH-induced immune responses and has partial cross-reactivity. In general, pathogenic vibrio parahaemolyticus carries at least one of tdh gene and trh gene which produce related toxins, or carries both virulence genes. Previously, colony-type food poisoning events have frequently occurred due to contamination with Vibrio parahaemolyticus, which has serotype O5: K17 and carries tdh toxin, or contamination with Vibrio parahaemolyticus, which has serotype O3: K6, carries tlh, tdh toxin.

At present, antibiotics are mainly used for preventing and treating vibrio parahaemolyticus, but a large amount and irregular use of antibiotics have caused serious drug resistance problem. In investigation of the resistance to Vibrio parahaemolyticus, many isolates were found to be resistant to streptomycin and ampicillin, and tdh and trh genes were found to be present in part of the isolates, which genes are involved in the pathogenesis of Vibrio parahaemolyticus infection. According to the assessment published by the canadian academy of sciences (CCA) 2019, by 2050, nearly 40% of bacterial infections will develop resistance to drugs now used to treat them. Resistance is expected to cause premature death of 1000 tens of thousands worldwide, estimated to be lost in the hundreds of trillion dollars each year.

The phage is used as a natural bacteriostat, and can effectively inhibit and lyse pathogenic bacteria. In the background of increasingly severe pathogen resistance problems, the limitation of antibiotic treatment is increasingly prominent, and phage as a potential biological control agent becomes an important choice for replacing antibiotics. Currently, phages are used more in clinical symptomatic treatment, and many patients infected with multidrug-resistant pathogens have significantly reduced symptoms after receiving phage treatment. In addition, the research and application of phage are more and more rich for common food-borne pathogenic bacteria such as salmonella, escherichia coli and the like, staphylococcus aureus, vibrio parahaemolyticus and the like. In the control of vibrio parahaemolyticus, phages have become a biological control agent candidate for replacing antibiotics. Phage VPp1 reduced Vibrio parahaemolyticus in oyster at 16℃at 0.1MOI by 2.35-2.76log CFU/g over 36 hours. The use of phage VPT02 (multiplicity of infection 10) increased the survival rate of saline shrimp infected with Vibrio parahaemolyticus from 16.7% to 46.7%, when applied to ready-to-eat food raw fish sections, VPT02 reduced Vibrio parahaemolyticus by up to 3.9 log compared to untreated phage controls. In view of the prevention and treatment of multi-drug resistant vibrio parahaemolyticus, further expansion of the application research on phage is still required.

Disclosure of Invention

Aiming at the defects existing in the prior art, the invention aims to provide a novel vibrio parahaemolyticus phage vB_ VpS _CC6 and application thereof. The invention evaluates the application effect of the whole genome and the biological characteristics of the whole genome in preventing and treating vibrio parahaemolyticus in salmon by analyzing the whole genome and forming phage cocktail, and further explores the potential of the whole genome in preventing and treating vibrio parahaemolyticus.

In order to achieve the above object, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a novel virulent vibrio parahaemolyticus phage vB_ VpS _CC6 (hereinafter abbreviated as CC 6) deposited at 9.14.2020 with the collection of microorganism strains in Guangdong province under the accession number GDMCC NO:61189-B1, under the taxonomic designation Vibrio Parahaemolyticus bacteriophage, and under the accession number Guangzhou city martyr, no. 100, college of university 59, building 5.

The vibrio parahaemolyticus phage is obtained by collecting and separating in a water sample of yellow sand aquatic product trading market in Guangzhou of Guangdong, the GenBank accession number of the phage nucleotide sequence is OK625546.1, and the phage has good temperature tolerance (4-60 ℃) and pH tolerance (pH 4-11) and certain ultraviolet tolerance (ultraviolet irradiation is less than 20 min), and can play a bacteriostatic role under different temperature, acid-base and ultraviolet irradiation time conditions.

In a second aspect, the present invention provides the use of phage CC6 as described above in the preparation of a biological antibacterial agent having an effect of inhibiting or killing Vibrio parahaemolyticus.

Preferably, the vibrio parahaemolyticus is multi-drug resistant vibrio parahaemolyticus.

Preferably, the multiple drug resistance comprises resistance to two or more antibiotics among ampicillin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, and streptomycin.

In a third aspect, the invention provides an application of the vibrio parahaemolyticus phage CC6 in preparing related medicines for preventing and treating diseases caused by vibrio parahaemolyticus infection. The medicine is used for human or animals to prevent and treat diseases of vibrio parahaemolyticus infection, such as prawn hepatopancreatic necrosis caused by vibrio parahaemolyticus, vibriosis and other related diseases.

In a fourth aspect, the invention provides an application of the vibrio parahaemolyticus phage CC6 in preventing and treating aquatic product pollution caused by vibrio parahaemolyticus.

In a fifth aspect, the present invention provides a bactericidal or pharmaceutical composition comprising the above-mentioned vibrio parahaemolyticus phage CC6 as an active ingredient.

Preferably, the composition further comprises other co-formulated bactericidal active ingredients or adjuvants.

Preferably, the other matched bactericidal active ingredients include phages BA3, BA6, CA8, FE11 and G1. The composition provided by the invention can selectively comprise a plurality of phages to form a phage cocktail combination, so that the host spectrum range is enlarged, and the inhibition effect on a plurality of vibrio parahaemolyticus is obviously increased through the synergistic effect among phages.

In a sixth aspect, the invention provides the use of the above composition for the preparation of a product for the prevention and treatment of infection by Vibrio parahaemolyticus.

Preferably, the product comprises a medicament, a disinfectant, a cleanser, a feed additive, or the like.

Preferably, the invention provides application of the composition in preparing a water disinfectant, wherein the water disinfectant is applied to cultivation, transportation and preservation of aquatic products such as fish, shrimp and crab.

Preferably, the invention also provides application of the composition in preparing aquatic animal feed additives.

Compared with the prior art, the invention has the beneficial effects that:

(1) The novel vibrio parahaemolyticus bacteriophage CC6 is separated, can crack multiple drug-resistant pathogenic bacteria, can crack 30% (17/55) drug-resistant vibrio parahaemolyticus tested, has a cracking effect on ampicillin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin and streptomycin antibiotic-sensitive vibrio parahaemolyticus, can replace antibiotics to play a bacteriostatic role to a certain extent, reduces antibiotic residues, provides a new scheme for solving the drug resistance problem faced by the current antibiotic treatment, and has good utilization value and application prospect.

(2) The vibrio parahaemolyticus phage CC6 provided by the invention has good temperature tolerance (4-60 ℃) and pH tolerance (pH 4-11) and certain ultraviolet tolerance (ultraviolet irradiation <20 min), and can play a role in bacteriostasis under different conditions of temperature, acid and alkali and ultraviolet irradiation time.

(3) The bacteriophage CC6 provided by the invention has relatively short incubation period, short antibacterial action time and quick effect on vibrio parahaemolyticus. Has good prevention and control effects on the pollution of aquatic products caused by vibrio parahaemolyticus.

(4) The phage CC6 provided by the invention can be continuously combined with other phages to prepare a composition, so that the problem that a single phage is easy to generate resistance is avoided, the bacteriostasis effect on vibrio parahaemolyticus is enhanced through the synergy among phages, the additive amount of a single phage under the same condition is reduced, the cost is saved, the host spectrum range is expanded, and the composition has an inhibition effect on various vibrio parahaemolyticus and can be used for broader-spectrum antibacterial or bacteriostatic application.

Drawings

FIG. 1 is a photograph of plaques of phage CC6 on a double-layered agar plate.

FIG. 2 is a protein functional map of phage CC 6.

FIG. 3 shows the results of phage CC6 VIRIDIC analysis.

FIG. 4 shows the results of the temperature stability measurement of phage CC 6.

FIG. 5 shows the pH stability measurement results of phage CC 6.

FIG. 6 shows the result of the ultraviolet tolerance test of phage CC 6.

FIG. 7 shows the results of a complex of optimal infection (MOI) assay for phage CC 6.

FIG. 8 is a one-step growth curve of phage CC 6.

FIG. 9 shows the results of a test of phage CC6 for killing Vibrio parahaemolyticus.

FIG. 10 shows the results of phage CC6 cocktail control experiments.

FIG. 11 shows the result of the control experiment of phage CC6 salmon.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The test methods used in the examples of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, are commercially available reagents and materials.

EXAMPLE 1 isolation culture of phages

(1) Activation and cultivation of host bacteria

The vibrio parahaemolyticus used in the invention is provided by the microorganism research institute of Guangdong province, a strain glycerol storage tube is taken out of a refrigerator, bacterial liquid is sucked into 3% sodium chloride alkaline peptone water, shake culture is carried out for 24 hours at 37 ℃ and 200r/min, the resuscitated bacterial liquid is taken out to line in a vibrio chromogenic plate, single bacterial colony is selected and inoculated into 5mL TSB liquid culture medium, and bacterial suspension is obtained after culture for 6-8 hours to logarithmic phase.

(2) Isolation and purification of phages

Collecting a water sample in a yellow sand aquatic product trade market sewer of Guangzhou province, centrifuging 5000 Xg of the water sample for 10min, vacuum-filtering supernatant with a 0.45 mu m filter membrane, adding anhydrous magnesium sulfate with a final concentration of 50mM into the filtrate, standing at room temperature for 10-20 min, vacuum-filtering the water sample with the 0.22 mu m filter membrane, and collecting the filter membrane. 90mL of eluent (3% beef extract, 3% Tween 80,5mM NaCl) was prepared, the collected filters were sheared and added to the eluent for ultrasound for 10min, then the eluent was filtered and sterilized in an ultra clean bench using a 0.22 μm filter, and the resulting filtrate was kept at 4℃for further use. 100. Mu.L of the log phase host bacterial suspension was aspirated, 2mL of the above treated filtrate was added, and the mixture was added to two-component TSB containing 2mM CaCl ₂, and after culturing at 37℃for 24 hours, the mixture was filtered through a 0.45 μm filter head to obtain phage culture broth. Using a double-layer plate method to identify whether phage filter culture broth contains virulent phage, 100. Mu.L of log phase bacterial broth was mixed with 5mL of TSB containing 0.4% soft agar (containing 2mM CaCl ₂), poured onto a 1.5% TSA plate, after the soft agar solidified, 2. Mu.L of phage broth was dropped, and after preparation was completed, placed at 37℃and after 8 hours of culture, the plate was observed for the formation of clear plaques. Spot picking purification was performed from the plaque-forming phage expansion broth by mixing 100. Mu.L of host bacteria and 100. Mu.L of diluted phage solution, adding 5mL of 0.4% TSA (containing 2mM CaCl ₂), culturing at 37℃for 8 hours, picking spots with a sterile gun head, then placing in 1mL of TSB (containing 2mM CaCl ₂) and culturing at 37℃for 24 hours, and then filtering the culture broth through a 0.22 μm filter head, thus obtaining a phage stock solution after one purification, and purifying 5-6 times by a double-layer plate method. Finally, the phage was obtained and named vB_ VpS _CC6 (hereinafter abbreviated as CC 6) and deposited with the Guangdong province microorganism strain collection center with the deposit number GDMCC NO:61189-B1.

EXAMPLE 2 identification of phage

(1) Morphology of phage on double-layered plates

100. Mu.L of log-phase host bacterial suspension and 100. Mu.L of phage with a certain dilution are mixed and added to 5mL of 0.4% TSA (containing 2mM CaCl ₂), poured onto a 1.5% TSA plate and cultured for 8 hours at 37 ℃, and then the obtained mixture can be observed and recorded. As a result, as shown in FIG. 1, phage CC6 produced plaques with a diameter of about 0.2mm, clear and transparent, and with halos at the edges.

(2) Phage DNA extraction, sequencing and genomic analysis

1ML of log phase host bacteria and 1mL of phage were added to a 50mL conical flask of TSB (2 mM CaCl ₂), and incubated at 37℃at 200rpm for 6h. After completion of the culture, 5000 Xg was centrifuged for 5min, 30mL of the supernatant was sucked and filtered through a 0.45 μm filter, 6mL of polyethylene glycol 8000 (60%) and 4mL of sodium chloride solution (5M) were added, and after gently stirring, the mixture was left at 4℃overnight. 12000 Xg was removed the next day, centrifuged at 4℃for 20min, and the resulting pellet was resuspended using 1mL SM buffer to give phage concentrate. The resulting 1mL phage concentrate was separated into two 1.5mL centrifuge tubes, and 32.5. Mu.L of DNA buffer and 5. Mu.L of DNaseI were added to each tube, followed by 5. Mu.L of RNase A at 37℃and a warm bath for 1h. After incubation, 20. Mu.L of 10% SDS, 20. Mu.L of EDTA, 1. Mu.L of proteinase K were added and incubated at 65℃for 1h. After the incubation, an equal volume of Tris saturated phenol solution was added, vortexed for 30s,12000 Xg, centrifuged for 5min, and the upper aqueous phase was transferred to a new centrifuge tube. An equal volume of phenol-chloroform-isoamyl alcohol mixture (25:24:1) was then added, vortexed for 30s,12000 Xg, centrifuged for 5min, and the upper aqueous phase was removed to a new centrifuge tube. The upper aqueous phase was extracted with an equal volume of chloroform and placed in a new centrifuge tube, and the chloroform extraction was repeated twice. Then adding equal volume of isopropanol, mixing, and standing at-20deg.C for 30min. After removal from the refrigerator, centrifugation was performed at 12000 Xg for 20min at 4 ℃. The pellet was collected, and the DNA pellet was washed with 200. Mu.L of 70% ethanol, 12000 Xg, and centrifuged for 5min (repeated twice). The ethanol is discarded and the ethanol is removed, and drying the DNA precipitate at room temperature for 5-10 min. 50. Mu.L of sterile water preheated at 65℃was added to dissolve the DNA sufficiently and the DNA was stored at-20℃for further use. Phage DNA was sequenced whole genome using Illumina MiSeq sequencing platform and spliced using SPAdes v.3.12.2 software. Subsequently, phage genome information is annotated by Prokka. The genome was visually analyzed using an online website CGview (http:// cgview. Ca /). The phage CC6 was analyzed for the presence of drug resistance genes and virulence factors using a database of drug resistance genes (https:// card. Mcmaster. Ca/home) and a database of virulence factors (http:// www.mgc.ac.cn/VFs /). The inter-genome similarity was analyzed using an on-line tool VIRIDIC (http:// rhea. Icbm. Uni-oldenburg. De/VIRIDIC /) tool. Phage vB_ VpS _CC6 is a vibrio phage belonging to the genus Mardecavirus of class Caudoviricetes, has linear double-stranded DNA, has a genome full length of 76917bp, and has a GenBank accession number of OK625546.1. As shown in FIG. 2, phage CC6 genome predicts a total of 106 Open Reading Frames (ORFs), 48 of which are similar to genes encoding known functional proteins (45.28%). The kit can be divided into five functional modules, namely DNA metabolism related proteins, phage structure related proteins, phage assembly related proteins, bacterial lysis related proteins and other functional proteins. Wherein, endolysin-related proteins are present in the lysis-related module, endolysin (Endolysin) is an enzyme encoded by phage, the main function being to lyse the cell wall of the host bacteria, releasing progeny phage particles. It plays a critical role in the phage lysis cycle. Through the searching and analysis of a virulence factor database and a drug-resistant gene database, the phage vB_ VpS _CC6 has no virulence factor and drug-resistant gene, and has application safety.

The VIRIDIC (Virus Intergenomic Distance Calculator) analysis of phage whole genome is a classification tool based on genome sequence similarity, which is used to determine the classification status (such as genus or species) of phage, and as can be seen from FIG. 3, the highest similarity between phage vB_ VpS _CC6 and PG288 (GenBank accession number: OQ 680630.1) is 96.2%. According to the genomic collinearity analysis, it was shown (DOI: 10.1016/j. Viruses res. 2024.199320) that for phage PG288, which shows the highest similarity to phage vB-VpS-CC 6, a rearrangement of some regions in both phage genomes occurred. Furthermore, the biological properties show that phage PG288 withstands temperatures between 20-50℃but phage CC6 survives at 60 ℃. The one-step growth curve shows that the PG288 latency is about 50 minutes, the lysis period is 100 minutes, 150 minutes later, and the lysis period is obviously different from the lysis period of phage CC6 latency of 10 minutes, 40 minutes later, which is the stability period. Thus, although phages CC6 and PG288 have similarities in their genomes, the difference between the biological properties also indicates that phage CC6 is a novel vibrio parahaemolyticus phage isolate, unlike PG 288.

Example 3 determination of phage vB_ VpS _CC6 biological Properties

(1) Phage CC6 detection of temperature, pH, UA stability

For the temperature tolerance experiment, 1mL of phage CC6 with a titer of 1X 10 ⁸ PFU/mL was taken, the phage was placed in a refrigerator at different temperature conditions (25, 37, 50, 60, 70 ℃) for 1h with treatment at 4 ℃) and equilibrated to room temperature after the completion, and the titer of phage was determined by the double-layer plate method. For the pH tolerance test, TSB broth was brought to different pH (ph=3, 4, 5, 6, 7, 8, 9, 10, 11, 12) with 1mol/L NaOH and 1mol/L HCl, and then filter sterilized using a 0.45 μm filter head. 0.1mL of 1X 10 ⁹ PFU/mL phage was added to 0.9mL of TSB broth of different pH and incubated at 37℃for 1h. After incubation, the phage titer was determined by the bilayer plate method by cooling to room temperature. For UV tolerance experiments, 5mL of 1X 10 ⁸ PFU/mL phage was added to a sterile petri dish. The disposable dish was left open under an ultraviolet lamp (254 nm,25 w) for about 15 cm. Sampling was started 0min, and samples were taken every 5min until all phages died. Finally, the titers of all samples were determined by a double-layer plate method. All the above 3 experiments were repeated three times.

As a result, as shown in FIG. 4, the phage CC6 titer was maintained at 1X 10 ⁸ PFU/mL or more in the range of 4℃to 50℃and was able to maintain its stability, and its titer was maintained at 1.53X 10 ⁶ PFU/mL at 60℃and was substantially inactivated to 70 ℃. As shown in FIG. 5, in the pH range of 5-11, phage CC6 titer was maintained at substantially around 1X 10 ⁸ PFU/mL, to pH3 and pH 12, and in the peracid or over-alkaline environment, phage protein capsids or nucleic acids were vulnerable, resulting in partial phage inactivation. As shown in FIG. 6, phage CC6 was sensitive to ultraviolet radiation, and the change in phage titer was evident substantially every 5 minutes, until phage CC6 had been killed after 20 minutes.

(2) Phage CC6 optimal multiplicity of infection (MOI) assay

Phage were diluted to 10 ¹⁰ to 10 ⁴ PFU/mL, host bacteria VP O3-11 was formulated to 1X 10 ⁸ CFU/mL, 100. Mu.L of phage of the corresponding titer and 100. Mu.L of host bacteria were added to 5mL of TSB (2 mM CaCl ₂) according to MOI 100,10,1,0.1,0.01,0.001,0.0001, respectively, and cultured at 37℃at 200rpm for 6 hours. The different cultures were sterilized by filtration through a 0.45 μm filter head. Subsequently, 100. Mu.L of phage solution was used to determine the titer by the double-layer plate method, and the experiment was repeated 3 times. The highest titre MOI is the optimal MOI.

MOI refers to the ratio of phage to the number of infected hosts during infection, phage are propagated at different MOIs, and phage titers also differ to find the optimal MOI, which is favorable for phage propagation at higher efficiency. As shown in FIG. 7, when the MOI was 10, 0.1, 0.01 and 0.001, there was no significant difference in the titer of phage CC6, indicating that the MOI was the optimal MOI of phage vB_ VpS _CC6, and that the good proliferation effect was achieved by using the MOI to proliferate phage vB_ VpS _CC6. At an MOI of 100, 0.0001, phage CC6 titers were lower, indicating that it is less suitable for proliferation at higher or lower MOI values.

(3) Phage CC6 one-step growth curve assay

Host bacteria cultured to logarithmic phase were taken 1X 10 ⁸ CFU,12000 Xg, centrifuged for 5min, resuspended in 1mL SMbuffer for precipitation, then 100. Mu.L of the above host bacteria SM buffer resuspension was taken and added to 0.8mL SM buffer (0.2 mM CaCl ₂) with 100. Mu.L of phage at MOI of 0.1, and incubated at 37℃for 10min. After the incubation was completed, 12000×g was centrifuged for 5min, the supernatant was discarded, the pellet was resuspended with 1mL of SM buffer, then 0.1mL of liquid was added to a 50mL centrifuge tube containing 9.9mL of TSB (2 mM CaCl ₂), a small amount of liquid was removed for filtration and plating, and at the same time, after removal of liquid, 50mL centrifuge tube was shake-cultured at 37 ℃ at 200rpm, and then samples were taken every 5min for plating titer by a double-layer plate method after filtration until 60min. The amount of lysis was calculated according to the formula, amount of lysis = average burst in stationary phase/amount of phage adsorbed into host bacteria.

The phage lysis period can be intuitively judged through a one-step growth curve, so that the incubation period and the lysis period of the phage are important indexes for evaluating the application potential of the phage, as shown in fig. 8, the phage mainly completes the adsorption process in the incubation period, the phage titer is generally low, the incubation period of phage CC6 is 0-10 min, the incubation period is relatively short, the lysis period is 10-40 min, a large number of progeny phage are released by the phage at the moment, the titer is obviously increased, the phage enters the stabilization period after 40min, and the lysis amount is 56PFU/cell.

(4) Phage CC6 lysis profile assay

The phage lysis profile was tested using 55 drug-resistant vibrio parahaemolyticus stored in this laboratory as host bacteria. The vibrio parahaemolyticus to be tested is respectively cultivated to the logarithmic phase, 100 mu L of bacteria liquid in the logarithmic phase is added into 5mL TSB containing 0.4% agar (containing 2mM CaCl ₂), the mixture is evenly poured into a 1.5% TSA flat plate, after the upper layer soft agar is solidified, 2 mu L of phage liquid is respectively added, after drying, the culture is carried out for 8 hours at 37 ℃, transparent plaques are observed, and whether the phage lyses the strain to be tested can be judged.

As shown in Table 1, phage CC6 was able to lyse 30% of the tested Vibrio parahaemolyticus (17/55), covering a total of 9 serotypes, including O1, O2, O3, O5, O6, O8, O9, O10, O11. Phage CC6 can infect mainly vibrio parahaemolyticus sensitive to ampicillin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin and streptomycin, and has no infection ability to the sensitivity of tetracycline and compound neonomine antibiotic resistance. Of 17 strains of Vibrio parahaemolyticus that can be infected, all are resistant to more than two antibiotics, wherein the strain resistant to ampicillin is up to 100%, ciprofloxacin is 64.71%, chloramphenicol is 17.65%, gentamicin is 29.41%, kanamycin is 35.29%, and streptomycin is 88.24%.

TABLE 1 phage vB_ VpS _CC6 lysis Spectrum

Note that +plaques were clear and transparent, -plaques free. Amp, ampicillin (ampicillin), c, chloramphenicol (chloramphenicol), cip, ciprofloxacin (ciprofloxacin), CN. gentamicin (gentamicin), k, kanamycin (KANAMYCIN), s, streptomycin (Streptomycin), TE. tetracycline (TETRACYCLINE), sxt, compound neonomine (Trimethoprim-Sulfamethoxazole).

Example 3 phage CC6 control experiment

(1) Test of phage CC6 for killing Vibrio parahaemolyticus VP O3-11

Host bacteria cultured to logarithmic phase were taken 1×10 ⁸ CFU, phage CC6 suspension was added, left standing for 15min at 37 ℃, then the mixture was placed at 37 ℃ for 200r/min culture, sampled every 2h and appropriately diluted and spread on TSA plates starting from 0h until 24h, the spread plates were left standing for 24h at 37 ℃ for colony counting, and experiments were performed in three independent replicates.

As shown in FIG. 9, vibrio parahaemolyticus O3-11 (original host) without phage was cultured for 2 hours, and the stable period was reached, increasing from 1X 10 ⁸ CFU/mL to about 1X 10 ⁹ CFU/mL, and the amount was maintained within 24. After the phage CC6 and VP O3-11 are co-cultured in the treatment group, the number of VP O3-11 is obviously reduced before 4 hours, and after 4 hours, the number of VP O3-11 is in a stable fluctuation state and is maintained at about 1X 10 ⁶ CFU/mL, and the phage CC6 obviously inhibits the growth of vibrio parahaemolyticus VP O3-11 and has good inhibition effect within 24 hours.

(2) Phage CC6 cocktail prevention and control experiment

(I) Phage CC6 cocktail host profile assay

Phage CC6 was prepared into phage cocktails by mixing phage CC6 with known phages BA3 (DOI: 10.3389/fmib.2020.00259), BA6 (DOI: 10.1007/s 00705-019-04351-5), CA8 (DOI: 10.3389/fmib.2020.00259), FE11 (DOI: 10.3390/v 14020264), and G1 (CN 116410935A), and then subjecting the 6 phages to cleavage spectrum measurement by adding them separately and adding them in equal proportions.

The results are shown in Table 2, and compared with the single phage lysis spectrum, the six phage combination lysis range is enlarged, and 147 strains to be tested can be lysed.

TABLE 2 phage cocktail and individual phage lysis spectra

(Ii) Phage CC6 cocktail prevention and control experiment

According to the results of the single-strain phage and cocktail host spectra in Table 2, single-strain phage combinations CC6, BA3, BA6, CA8, FE11 and G1 are respectively set, double-strain phage combinations CC6 and CA8, three-strain phage combinations CC6 and CA8 and FE11, four-strain phage combinations CC6 and CA8 and FE11 and BA3, five-strain phage combinations CC6 and CA8 and FE11 and BA6, six-strain phage combinations CC6 and BA3 and BA6 and CA8 and FE11 and G1, and a prevention and control experiment is carried out on vibrio parahaemolyticus VP O3-11. mu.L of log phase host bacterial broth and 100. Mu.L of phage were added to 5mL of TSB medium (2 mM CaCl ₂). After mixing well, 200. Mu.L was pipetted into a 96-well plate. A control of 200. Mu.L of TSB broth was used as a control with equal amounts of broth. After all additions were placed in an microplate reader (BioTek Instruments inc., winooski, VT, USA), the procedure was set for 37 ℃ incubation, measurement was carried out for 24 hours, starting with 0 hours, and OD ₆₀₀ was measured every hour. Experiments were repeated 3 times.

As shown in FIG. 10, among the individual phages, BA3 had the best control effect on VP O3-11, and the individual phages CC6, CC6+CA8 combination and CC6+CA8+FE11 combination could substantially inhibit VP O3-11 growth within the first 6 hours, and effectively delay the log phase. At 24h, the four-strain phage CC6+CA8+FE11+BA3 combination and the five-strain phage CC6+CA8+FE11+BA3+BA6 combination have equivalent effects, and the six-strain phage combination CC6+BA3+BA6+CA8+FE11+G1 has optimal prevention and control effects on VP O3-11, so that the OD ₆₀₀ of the six-strain phage combination is reduced by 32.64%, and the growth of VP O3-11 is effectively inhibited.

(3) Phage CC6 salmon prevention and control experiment

The purchased salmon sample is divided into small blocks of 1X 1cm ², washed three times with normal saline, placed in a disposable sterile culture dish, irradiated with ultraviolet lamp for 30min, turned over with sterile forceps, and irradiated with ultraviolet lamp for 30min. The treated salmon samples were transferred to 50mL centrifuge tubes, respectively. After the host bacteria are cultured to be properly diluted in the logarithmic phase, 100 mu L of bacterial liquid is added into a centrifuge tube, and the mixture is kept stand for 10min to be adsorbed. 100. Mu.L of phage CC6 was pipetted into the centrifuge tube at different MOI values, and samples with only bacterial liquid and no phage liquid were used as controls. 10mL of physiological saline was added to each of the treatment group and the control group for elution, and 100. Mu.L of the eluate was applied to a TSA plate after proper dilution, and the number of viable bacteria was measured. The samples were placed in a 4 ℃ refrigerator and viable counts were made for the samples at 0h, 3h, 6h, 9h, 12h, 24 h.

As a result, as shown in FIG. 11, in the salmon sample to which no phage was added, the amount of Vibrio parahaemolyticus was not reduced although it was not significantly grown, and it was found that the cryopreservation only inhibited the reproduction rate but did not kill the Vibrio parahaemolyticus contained therein under the condition of 4 ℃. When phage is added for prevention and treatment, when MOI=1000, phage CC6 has weak inhibition effect on vibrio parahaemolyticus VP O3-11, but when MOI=10000, the inhibition effect of CC6 on vibrio parahaemolyticus O3-11 is obviously enhanced, VP O3-11 is obviously inhibited, and when 24 hours, CC6 effectively eliminates 87.60% of VP O3-11, so that 3.14log ₁₀ CFU/block is reduced, and stronger antibacterial effect can be maintained. Studies have shown that treatment with phage mixtures on the surface of crayfish meat supplemented with multi-drug resistant Vibrio parahaemolyticus LVP1 resulted in a significant reduction in bacterial load of 2.36log ₁₀ CFU/mL. Furthermore, in the raw fish meat slices to which Vibrio parahaemolyticus is added, the use of phage VPT02 can reduce Vibrio parahaemolyticus therein by up to 3.9 log. Therefore, the phage CC6 has a strong antibacterial effect on vibrio parahaemolyticus O3-11 in salmon, and shows the potential of the phage CC6 as a bactericidal food additive.

It should be understood that the foregoing description of the specific embodiments is merely illustrative of the invention, and is not intended to limit the invention, and that any modifications, equivalents, improvements, etc. that fall within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims (9)

1. A novel Vibrio parahaemolyticus phage vB_VpS_CC6, characterized in that it was deposited on September 14, 2020 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 61189-B1, and the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. 2. The application of the novel Vibrio parahaemolyticus phage vB_VpS_CC6 according to claim 1, characterized in that the application includes: (i) Inhibits the growth and reproduction of Vibrio parahaemolyticus; (ii) Kill Vibrio parahaemolyticus; (iii) To prepare drugs for treating diseases caused by Vibrio parahaemolyticus infection; (iv) Preventing and controlling aquatic product contamination caused by Vibrio parahaemolyticus. 3. The application according to claim 2, wherein the Vibrio parahaemolyticus is a multidrug-resistant Vibrio parahaemolyticus. 4. The application according to claim 3, wherein the multidrug resistance refers to resistance to two or more antibiotics among ampicillin, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, and streptomycin. 5. The application according to claim 2, characterized in that the drug is used on humans or animals to prevent and treat diseases caused by Vibrio parahaemolyticus infection, including shrimp hepatopancreatic necrosis and vibriosis. 6. An antibacterial composition, characterized in that its active ingredient comprises the Vibrio parahaemolyticus phage vB_VpS_CC6 as described in claim 1. 7. The composition according to claim 6, characterized in that it further comprises a synergistic antibacterial active ingredient or adjuvant, said synergistic antibacterial active ingredient being composed of bacteriophages BA3, BA6, CA8, FE11 and G1. 8. Use of the composition according to claim 6 or 7 in the preparation of products for preventing and treating Vibrio parahaemolyticus infection. 9. The application according to claim 8, wherein the product comprises disinfectants, feed additives, drugs, and cleaning agents.