CN110093321B

CN110093321B - Application of bacteriophage AH10-Phage-QY01 in preparing medicines for treating or preventing and controlling aquaculture bacterial diseases

Info

Publication number: CN110093321B
Application number: CN201910361627.2A
Authority: CN
Inventors: 吕利群; 徐宁; 王浩
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2019-04-30
Filing date: 2019-04-30
Publication date: 2021-07-13
Anticipated expiration: 2039-04-30
Also published as: CN110093321A

Abstract

The invention discloses application of aeromonas hydrophila Phage AH10-Phage-QY01 in preparation of a medicine for treating or preventing and controlling aquaculture bacterial diseases, and belongs to the technical field of prevention and control of aquaculture bacterial diseases. The bacteriophage AH10-Phage-QY01 is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2019109, and the application of the bacteriophage AH10-Phage-QY01 in preparing bacterial septicemia diseases caused by aeromonas hydrophila can be used independently or combined with antibiotics. The invention utilizes the universality of the existence of the Phage and the high specificity to host bacteria, screens out Aeromonas hydrophila Phage AH10-Phage-QY01 for the first time from the natural environment aiming at pathogenic bacteria, is used for inhibiting and cracking the pathogenic bacteria, and treats or prevents and controls bacterial fish diseases such as bacterial septicemia and the like caused by the pathogenic bacteria.

Description

Application of bacteriophage AH10-Phage-QY01 in preparing medicines for treating or preventing and controlling aquaculture bacterial diseases

Technical Field

The invention belongs to the technical field of prevention and treatment of aquaculture bacterial diseases, and particularly relates to aeromonas hydrophila Phage AH10-Phage-QY01 and application thereof in preparation of a medicine for treating or preventing and controlling aquaculture bacterial diseases.

Background

Bacterial fish diseases are the main diseases of fresh water aquaculture industry, and not only cause large-area death of cultured species and fish eggs during the epidemic period, but also cause the loss of commodity value of cultured products and serious economic loss. Bacterial fish diseases are easy to get and difficult to treat and easily generate drug resistance, and at present, the prevention and control difficulty of bacterial fish diseases is increased due to the fact that a large amount of antibiotic drugs are used and the drug resistance of various aquatic pathogenic bacteria is increased.

Septicemia caused by Aeromonas Hydrophila (AH) infection is a highly pathogenic bacterial disease of carp and crucian, the pathogen of the septicemia is Aeromonas Hydrophila (AH), bacterial septicemia occurs frequently, and destructive influence is caused on crucian breeding industry. Aeromonas (Aeromonas) belongs to Brevibacterium, monarda, facultative anaerobic gram-negative bacteria with extremely active movement, wide growth temperature (0-45 ℃) and low nutritional requirement, a common flat plate and a slightly white semitransparent bacterial colony form a gray, smooth, moist and convex bacterial colony on blood agar, most of the bacterial colonies have beta hemolysis, most of the bacterial colonies form lactose nonfermentation bacterial colonies on an intestinal tract selection culture medium, and the growth on TCBS agar is poor. The aeromonas hydrophila infects crucian to cause extensive bleeding of internal organs, gills and body surfaces, the disease is generally violent, the crucian is a malignant infectious disease, and the death rate is extremely high.

The conventional antibacterial drugs of aeromonas hydrophila such as thiamphenicol, gentamicin, terramycin, sulfamethoxazole and the like exist, and the main components of the conventional antibacterial drugs are antibiotics or sulfonamides. Because of the long-term use of a large amount of antibiotics, the drug resistance of bacteria to the antibiotics is enhanced year by year, the bacteriostatic effect of the antibiotics is reduced year by year, the dosage of the antibiotics required for achieving the treatment effect is increased, and the situation that strong-drug-resistant bacteria such as aeromonas hydrophila and the like are available without drugs is faced for a long time.

Phage (Phage) is a virus that attacks bacteria, must be parasitic in the living bacteria, and has strict host specificity, which depends on the molecular structure and complementarity of the Phage-adsorbed organ and the receptor on the surface of the recipient bacterium. Bacteriophages are the most prevalent and widely distributed group of viruses. Bacteriophages are usually found in places which are filled with bacterial flora, such as soil, in the intestine of animals. By utilizing the universality of the existence of the phage and the high specificity to host bacteria, the corresponding phage can be screened for the pathogenic bacteria of the aeromonas hydrophila and can be used for inhibiting and killing the pathogenic bacteria. The method of using the phage or combining the phage and the antibiotic to replace the simple antibiotic therapy can effectively reduce the drug resistance of the bacteria to the antibiotic. Meanwhile, because the bacteriophage generally exists in the environment, the bacteriophage therapy does not greatly affect the environment, and is green, safe and reliable.

Disclosure of Invention

The invention utilizes the universality (namely the safety, the Phage is widely existed in the nature and can not generate adverse effect on the environment) of the existence of the Phage and the high specificity to host bacteria, screens out Aeromonas hydrophila Phage AH10-Phage-QY01 from the natural environment for the first time aiming at pathogenic bacteria, and can be used for inhibiting and cracking the pathogenic bacteria and treating or preventing and controlling bacterial fish diseases such as bacterial septicemia and the like caused by the pathogenic bacteria.

Aiming at the problems that septicemia caused by bacterial fish diseases, particularly Aeromonas Hydrophila (Aeromonas Hydrophila) infection, is easy to get and difficult to treat, has strong drug resistance and has high prevention and control difficulty at present, the invention aims to provide the application of Aeromonas Hydrophila Phage AH10-Phage-QY01 in preparing the drugs for treating or preventing and controlling aquaculture bacterial diseases.

The technical scheme adopted by the invention for solving the technical problems is as follows:

aeromonas hydrophila Phage AH10-Phage-QY01(Aeromonas hydrophylla bacteriophages AH10-Phage-QY01) is preserved in the China center for type culture collection in Wuhan university, Wuhan City, Hubei, 2.25.2019 with the collection number of CCTCC NO: M2019109.

The application of the aeromonas hydrophila Phage AH10-Phage-QY01 in preparing medicines for treating or preventing and controlling bacterial diseases of aquaculture.

Further, the medicine takes Aeromonas hydrophila Phage AH10-Phage-QY01 as an active ingredient, and also comprises a pharmaceutically acceptable carrier; the Aeromonas hydrophila Phage AH10-Phage-QY01 is used at a dosage of not less than 1 × 10⁷pfu/mL. Further, the bacteriophage or the lysate thereof is put into the aquaculture water body, so that the quantity of the bacteriophage in the aquaculture water body reaches 1 multiplied by 10 to the minimum⁷pfu/mL～9×10⁷pfu/mL (typically, the titer of the phage stock solution prepared is about 10)¹²pfu/mL, i.e. dosing stock solution at 1: 100000 dilution) and can play a better control role on aeromonas hydrophila in the water body.

Further, the application of the aeromonas hydrophila Phage AH10-Phage-QY01 in preparing a medicine for treating bacterial septicemia caused by aeromonas hydrophila.

Further, the Aeromonas hydrophila Phage AH10-Phage-QY01 was used alone or in combination with antibiotics.

Further, the Aeromonas hydrophila Phage AH10-Phage-QY01 was used alone at a dose of not less than 1X 10⁷pfu/mL; the dosage ratio of the aeromonas hydrophila Phage AH10-Phage-QY01 in combination with antibioticsIs 1: 1, and the aeromonas hydrophila Phage AH10-Phage-QY01 is used in a dosage of not less than 1 × 10⁷pfu/mL. AH 10-phase-QY 01 can be used as an antibiotic adjuvant therapy agent, plays a role in preventing and controlling pathogenic bacteria with drug resistance when being combined with antibiotics, has the same using dosage with the antibiotics and the single using dosage, and has the dosage ratio of 1: 1.

the invention has the beneficial effects that:

1. compared with the traditional drug therapy of aquatic pathogenic bacteria, the method utilizes the universality of Phage and high specificity to host bacteria, screens out Aeromonas hydrophila Phage AH10-Phage-QY01 from the natural environment for the pathogenic bacteria for the first time, is used for inhibiting and cracking the pathogenic bacteria, and treats or prevents and controls bacterial fish diseases such as bacterial septicemia and the like caused by the pathogenic bacteria.

2. The screened aeromonas hydrophila Phage AH10-Phage-QY01 has very strong bacteriostatic effect on aeromonas hydrophila and long duration, and the Phage can have good bacteriostatic effect on aeromonas hydrophila with strong drug resistance by replacing antibiotics or combining the antibiotics, is used for preventing and treating aquaculture aeromonas hydrophila disease and reducing the using amount of the antibiotics.

3. The screened aeromonas hydrophila Phage AH10-Phage-QY01 has high specificity, has good inhibition effect on the aeromonas hydrophila without influencing the growth of other colonies, and is green, safe and efficient.

Drawings

FIG. 1 shows the bacteriostatic effect of Aeromonas hydrophila Phage AH10-Phage-QY01 on Aeromonas hydrophila; wherein the multiplicity of infection is 0.1, and the culture is carried out at 37 ℃ for 8 h.

FIG. 2 shows duration of the bacteriostatic effect of Aeromonas hydrophila Phage AH10-Phage-QY01 on Aeromonas hydrophila; wherein, the multiplicity of infection is 0.1, and from left to right in sequence: culturing at 37 degrees for 12h, culturing at 37 degrees for 24h, and culturing at 37 degrees for 48 h.

The preservation information is as follows:

Detailed Description

The invention is further illustrated with reference to the following figures and examples.

Example (b): screening, separation and purification of Aeromonas hydrophila Phage AH10-Phage-QY01

By utilizing the universality of existence of the phage and the high specificity to host bacteria, in the following examples, various aquaculture pathogenic bacteria are targeted, and the corresponding phage are screened from the natural environment and used for treating aquaculture bacterial diseases caused by the pathogenic bacteria. The method comprises the following specific steps:

a sample of 50ml is taken and centrifuged at 4000rpm/min for 30 min.

10ml of the supernatant was aspirated and sterilized by filtration through a 0.22um filter.

Adding 25ml of LB liquid culture medium concentrated by 3 times into a 200ml conical flask, cooling to room temperature, adding the filtrate into the conical flask, adding 1ml of host bacteria, simultaneously adding 0.5M calcium chloride to a final concentration of 0.1mM, and culturing at 28 ℃ for 12-14 h.

Taking out 3ml of the culture solution, adding into a 5ml sterile test tube, adding chloroform to a final concentration of 3%, shaking for 2min, and standing at room temperature for 2 h.

Filtering 1ml of supernatant with 0.22um filter for sterilization, and diluting the filtrate 10 times to 10 times^-5。

Mixing 100ul of each gradient diluent with 200ul of host bacteria uniformly, incubating at 28 ℃ for 15min, mixing with 5ml of 42-DEG melted upper layer LB (0.4% agar), pouring on a bottom layer LB flat plate, standing at room temperature for 15min, and after the upper layer agar is solidified, inversely placing in a 28-DEG incubator for culturing for 8 h.

The plates cultured for 8h were observed, and a single spot was picked up in 5ml of LB liquid, 200ul of fresh host bacteria was added, calcium chloride was added to a final concentration of 0.1mM, and the plates were left to stand at 28 ℃ for overnight culture.

Taking out, centrifuging at 4000rpm/min for 30min, and filtering with 0.22um filter membrane to obtain filtrate, which is diluted by 10 times in gradient.

Repeating the steps of 6, 7 and 8 for 3-5 times until the plaque with stable shape, size and property is obtained.

And (3) picking the purified plaques, putting the plaques into a 1.5ml centrifuge tube containing 1ml of host bacteria, and culturing for 6-12 h at 37 ℃.

Centrifuging at 15000g for 10min, collecting supernatant, filtering with 0.22um filter membrane, adding into a new 1.5ml centrifuge tube, and adding 200ul PEG8000 solution.

Mixing by inversion, and standing for 15 min.

The phage particles were obtained by centrifugation at 15000g for 20 min. Resuspend with 200ul TE buffer to obtain high titer phage stock solution, which can be used for seed preservation or DNA sample extraction material.

Effect example 1: verifying the bacteriostatic effect of the phage

Taking Aeromonas hydrophila as an example, Aeromonas hydrophila Phage AH10-Phage-QY01 stock solution (the titer is 10) obtained by purifying and enrichment culturing in the example^-10～10^-12pu f/ml) was mixed with Aeromonas hydrophila at a multiplicity of infection of 0.1(20ul phage stock mixed with 200ul Aeromonas hydrophila), the double-layered plate was inverted after being placed in an incubator at 37 ℃ for 10 minutes, the plate was sealed with a sealing film and placed in the incubator at 37 ℃ for 8 hours, and then significant plaques were observed, as shown in FIG. 1.

Effect example 2: verifying duration of bacteriostatic effect of phage

As shown in FIG. 2, Aeromonas hydrophila Phage AH10-Phage-QY01 was able to rapidly and efficiently lyse Aeromonas hydrophila and inhibit its colony growth, and plaques appeared on the plate, which grew up with time until all plaques were joined together to complete the lawn on the plate.

Claims

1. Aeromonas hydrophila Phage AH10-Phage-QY01(Aeromonas hydrophila bacteriophages AH10-Phage-QY01) with a preservation number of CCTCC NO of M2019109, and the collection number is preserved in the China center for type culture collection in Wuhan university, Wuhan City, Hubei province in 2019 at 25.2.month.

2. Use of Aeromonas hydrophila Phage AH10-Phage-QY01 as claimed in claim 1 for the preparation of a medicament for the treatment or prevention of aquacultureUse of a germ disease drug comprising as an active ingredient said aeromonas hydrophila Phage AH10-Phage-QY01 in an amount of not less than 1 × 10⁷pfu/mL。

3. Use according to claim 2, wherein the use of aeromonas hydrophila Phage AH10-Phage-QY01 in the manufacture of a medicament for the treatment of bacterial sepsis disease caused by aeromonas hydrophila, said aeromonas hydrophila Phage AH10-Phage-QY01 used alone, or said aeromonas hydrophila Phage AH10-Phage-QY01 in a dose ratio to antibiotics of 1: 1, the combination is used.