CN111363723A

CN111363723A - Novel vibrio cholerae bacteriophage and application thereof

Info

Publication number: CN111363723A
Application number: CN202010169223.6A
Authority: CN
Inventors: 潘强; 任慧英; 孙虎芝; 闫艳新; 丁同燕
Original assignee: Qingdao No Antibiotics Biotechnology Co ltd
Current assignee: Qingdao No Antibiotics Biotechnology Co ltd
Priority date: 2020-03-12
Filing date: 2020-03-12
Publication date: 2020-07-03
Anticipated expiration: 2040-03-12
Also published as: CN111363723B

Abstract

The invention belongs to the field of biotechnology, and discloses a novel Vibrio cholerae bacteriophage and application thereof, wherein the latin name of the bacteriophage is Vibrio cholerae phase, the bacteriophage is named as vB-Vchs-PR02, the bacteriophage has broad-spectrum bactericidal capability on Vibrio cholerae, and is preserved in China general microbiological culture Collection center (CGMCC NO. 18861) in 2019 and 11 months and 04 days, and the preservation number is CGMCC NO. 18861. The bacteriophage has strong cracking effect on vibrio cholerae, can be used for preparing medicaments for preventing and treating vibrio cholerae infection or biological food therapy additives and disinfectants, and provides a safe and effective method for preventing and treating vibrio cholerae infection diseases.

Description

Novel vibrio cholerae bacteriophage and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a novel vibrio cholerae bacteriophage and application thereof.

Background

The Penaeus vannamei (Penaeus vannamei) is one of three excellent varieties with the highest yield in the world aquaculture industry, and has the characteristics of strong adaptability, high growth speed, strong disease resistance and the like. In the 80 s of the 20 th century, the development of the prawn aquaculture industry in China drives the rapid development of the aquaculture industry in China and promotes the development of economy in coastal areas in China. However, with the rapid increase of the culture scale and culture density of the penaeus vannamei boone in recent years, the culture water body gradually worsens, and the disease problem also becomes more serious.

Vibrio cholerae (Vibrio cholerae) is a common marine pathogen. Infection of Penaeus vannamei Boone with Vibrio cholerae is an important cause of white feces syndrome. The white feces syndrome has become a serious disease next to early death syndrome in the breeding production of the penaeus vannamei boone in recent years, and farmers talk about white feces color change, which causes the penaeus vannamei boone to die in a large amount. The pathogenesis of the white feces syndrome of the penaeus vannamei caused by the vibrio cholerae is probably that the vibrio cholerae grows and proliferates in the intestinal tissues of the penaeus vannamei, adheres to the surface of intestinal mucosa, generates virulence factors and causes intestinal wall mucosa injury, further leads to the hepatopancreas tissues through the haemolymph circulation and is propagated and generates toxin in the hepatopancreas tissues, so that the hepatopancreas is subjected to swelling, erosion and other pathological changes, finally promotes the synthesis and secretion function of the hepatopancreas and intestines to related digestive juice or digestive enzymes, and leads to the death of the penaeus vannamei.

At present, the traditional method for preventing and treating white feces syndrome of penaeus vannamei boone is western medicine antibiotics, but the large amount of antibiotics is used, so that water is polluted, pathogenic bacteria can generate drug resistance, the drug resistance is easy to generate due to the existence of various drug resistance mechanisms of vibrio cholerae, and the expected effect is difficult to achieve due to the use of the antibiotics. Therefore, the research and development of a novel preparation with obvious curative effect and safe use for killing the vibrio cholerae in the water body is very important for the prawn breeding industry.

The bacteriophage is also called bacterial virus, is a virus for specifically cracking bacteria, is widely distributed in the environment, has various types and simple structure, and has strong host specificity. Once the phage infects a bacterium, the phage proliferates within the bacterial cell. After proliferation, the progeny phage can destroy the cell wall of the bacteria to crack the host bacteria, and the phage is released after the host bacteria are cracked to infect new host bacteria, which shows that the phage has high-efficiency bacteria killing capability. Bacteriophage kills bacteria with high specificity, and a certain bacteriophage infects only a certain bacterium without harming others. The bacteriophage is utilized to control aquatic pathogenic bacteria and is applied before the bacteriophage is found, but the research on the bacteriophage is not sufficient, and the antibiotic has broad-spectrum bacteriostatic effect at that time, and has good curative effect and quick response, so that the application of the bacteriophage is greatly weakened. In recent years, due to a series of drug-resistant bacteria problems caused by abuse of antibiotics, phages regress to the visual field of people, and research on application of the phages to control pathogenic bacteria also has attracted wide interest in the scientific community. As one of the important weapons against antibiotic resistance, bacteriophage has great potential for clinical application.

However, no effective vibrio cholerae bacteriophage exists at present, which can be used for preventing and treating white feces syndrome of penaeus vannamei boone, so that the prior art needs to be further improved.

Disclosure of Invention

Aiming at the problems, the invention provides a novel vibrio cholerae bacteriophage and application thereof as a medicine and water body disinfectant, and the vibrio cholerae bacteriophage can be applied to the penaeus vannamei boone in a sprinkling and mixing manner, so that the effect of preventing and treating the white feces syndrome is achieved, and the difficult problem of medicine resistance in the process of treating the white feces disease of the penaeus vannamei boone by antibiotics can be avoided.

In a first aspect, the application provides a Vibrio cholerae bacteriophage which is separated from large-market seafood sewage and has broad-spectrum bactericidal capacity on Vibrio cholerae, has a latin name of Vibrio cholerae phase, is named as vB-Vchs-PR02, is preserved in China general microbiological culture Collection center on 11-month-04 in 2019, and has the preservation number of CGMCC NO. 18861.

The bacteriophage vB-Vchs-PR02 has strong cracking effect on vibrio cholerae, and provides a bacteriophage source for industrial production of safe and effective water disinfectants and medicines for preventing and treating white feces of penaeus vannamei caused by vibrio cholerae.

In a second aspect, the invention also provides application of the novel vibrio cholerae bacteriophage vB-Vchs-PR02 in medicines and water disinfectants for preventing and treating vibrio cholerae infection diseases.

Preferably, in the application, the disease is the white feces syndrome of the Penaeus vannamei Boone, the bacteriophage is used as a medicine of a medicinal active component or is added into the feed of the Penaeus vannamei Boone as a feed additive, has an obvious inhibiting and killing effect on Vibrio cholerae in the Penaeus vannamei Boone body, and can effectively prevent the occurrence of the white feces disease of the Penaeus vannamei Boone caused by the Vibrio cholerae. In addition, the bacteriophage can be directly sprayed into water body for administration.

In a third aspect, the invention also provides a medicine for preventing and treating diseases caused by vibrio cholerae infection, and the active component of the medicine comprises the novel vibrio cholerae bacteriophage vB-Vchs-PR 02.

In addition, the invention also provides a detection kit which comprises the novel vibrio cholerae bacteriophage. Those skilled in the art can prepare a detection kit by utilizing the vibrio cholerae bacteriophage vB-VchS-PR02 according to the disclosure of the invention and the common knowledge in the field, and the detection kit is used for detecting the specific infected vibrio cholerae and is used for detecting and controlling diseases caused by the infection of host bacteria vibrio cholerae.

The medicine also comprises auxiliary materials; the auxiliary material is one or more of SM buffer solution, sodium alginate, sucrose, maltodextrin and glucose. Preferably, the germicidal composition further comprises bacteriophages of specific pathogenic bacteria of different species of bacteria.

Specifically, the dosage form of the phage composition is powder, aqua, lyophilized agent, gel, cream or ointment.

In a fourth aspect, the invention also provides a water disinfectant, the effective component of which is the vibrio cholerae bacteriophage; preferably, the concentration of phage is 10⁶PFU/ml or more.

Further, the water disinfectant also comprises bacteriophage of other different kinds of specific pathogenic bacteria.

The invention has the following beneficial effects:

1. the invention provides a novel vibrio cholerae bacteriophage and application thereof, the bacteriophage has strong cracking effect on vibrio cholerae and wide cracking spectrum, and provides a bacteriophage source for industrial production of the bacteriophage and application of the bacteriophage in environmental disinfection and bacteriophage prevention and treatment of white feces disease of penaeus vannamei caused by vibrio cholerae.

2. The novel vibrio cholerae bacteriophage is convenient for industrial production and can be specifically propagated through host bacteria vibrio cholerae. The bacteriophage is also used for preparing safe and effective biological disinfectant, and is used for disinfecting the water environment so as to treat the animal breeding environmental pollution; the bacteriophage can also be used for preparing a safe and pollution-free novel medicine for preventing and treating white feces disease of Penaeus vannamei caused by Vibrio cholerae.

Drawings

FIG. 1 is a diagram of the morphology of bacteriophage vB-Vchs-PR02 observed under an electron microscope;

FIG. 2 shows the results of the thermostability of bacteriophage vB-Vchs-PR 02;

FIG. 3 shows the results of pH stability of bacteriophage vB-Vchs-PR 02;

FIG. 4 is a graph showing the effect of phage vB-VchS-PR02 on killing R17.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. In the present invention, the equipment and materials used are commercially available or commonly used in the art, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.

EXAMPLE 1 isolation culture of phage

Recovery culture of bacterial strain and preparation of bacterial suspension

And (3) selecting the cryopreserved bacterial liquid of the vibrio cholerae, streaking the cryopreserved bacterial liquid on a TCBS (TCBS) plate in three zones, separating a single bacterial colony, and culturing the single bacterial colony in a 37-DEG C incubator for 16-24 hours. A single colony was picked and inoculated into 5ml2216E broth, and cultured in an air shaker at 37 ℃ for 16h with shaking at 170rpm to obtain a single bacterial suspension.

(II) isolation and purification of bacteriophages

A water sample of a certain shrimp pond for cultivation in Binzhou city, Shandong province is taken and put into a sampling bottle containing 2216E broth, each strain of vibrio cholerae is added into each sampling bottle according to the amount of 0.5 percent, after the mixture is uniformly stirred, the mixture is subjected to shaking cultivation overnight at 170rpm in an air oscillator at 37 ℃, the mixture is centrifuged at 10000rpm for 10min, and a bacterial filter with the diameter of 0.22 mu m is used for filtration and sterilization. Mixing the filtrate and host bacteria, incubating for 5min at 37 ℃, adding the mixed solution into the melted upper agar, quickly rubbing the test tube by hands to mix the agar uniformly, then quickly pouring the test tube onto the prepared lower flat plate, slightly shaking the test tube to distribute the test tube uniformly, putting the test tube into a thermostat at 37 ℃ after solidification, and observing the result after 6-8 h. If the bacteriophage exists, transparent and regular circular plaques are formed on the culture medium, namely the plaques are formed. And (3) digging single plaques, centrifuging the plaques for 5min at 11000rpm in 1ml of PBS in an air oscillator at 37 ℃ in an air oscillator, taking the supernatant, obtaining the single plaques by using a double-layer plate method, and repeating the steps for 3-5 times until the plaques with consistent sizes and shapes are obtained.

Example 2 determination of biological characteristics of bacteriophages

(I) observing the form of the phage by a transmission electron microscope

Is taken to be higher than 1 × 10⁸Mu.l of the PFU/ml phage sample was dropped onto a microporous copper mesh, precipitated for 15min, and excess liquid was blotted off with filter paper. 15 μ l of 2% phosphotungstic acid (PTA) was dropped on the copper mesh, dyed for 5min, and excess dye solution was sucked off with filter paper, dried, observed with a transmission electron microscope and photographed.

The head of the phage is in a polyhedral structure as observed by an electron microscope, the diameter of the head is about 60nm, the tail is about 180-190 nm long, the form of the phage accords with the characteristics of the long-tail phage family according to the definition of the International Committee for Virus Classification (ICTV), and the phage belongs to the long-tail phage family and is named as vB-Vchs-PR 02.

(II) detection of thermostability of bacteriophage

1 × 10⁹The PFU/ml bacteriophage vB-VchS-PR02 proliferation solution is respectively acted for 20min, 40min and 60min in water bath at 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃, and each temperature is provided with two parallel groups. The titer of the phage was determined by the double-layer plate method.

The result shows that the phage vB-Vchs-PR02 basically keeps the original activity after 1h of action at 40 ℃ and 50 ℃ and still keeps at 1.0 × 10 after 1h of action at 60 DEG C⁸PFU/ml or more; the phage was substantially inactivated at a high temperature of 70 ℃ for 20 min. Thus, the bacteriophage vB-Vchs-PR02 has higher thermal stability.

(III) detection of pH stability of phage

Adding PBS 4.5ml with different pH values (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) into sterile test tubes, placing three tubes in a water bath kettle at 37 deg.C, and adding 500 μ l of 1 × 10 after temperature is stabilized⁹PFU/ml phage proliferation liquid, mixing uniformly at 37 deg.C in water bath for 1h, 2h, 3 h. After the reaction is finished, the pH value of the mixed solution is about 7 by adding a proper amount of HCl or NaOH into the mixed solution, and the titer of the phage is measured by a double-layer plate method.

As a result, the titer of the phage vB-Vchs-PR02 is hardly changed or slightly reduced within the pH range of 4-11 and still remains at 10⁸The pH value of the phage is wider than PFU/ml.

(IV) determination of the optimal multiplicity of infection (MOI) of the phages

The vibrio cholerae phage vB-VchS-PR02 and the host bacterium R17 are propagated according to a conventional method, the titer of the phage and the host bacterium is measured, and the phage vB-VchS-PR02 and the host bacterium R17 are appropriately diluted. 100 μ l each of vB-Vchs-PR02 and R17 was added to 2216E broth at a multiplicity of infection of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0.000001. The culture was shaken at 37 ℃ and 170rpm until the liquid became clear and the shake-up time was recorded. Centrifuging at 11000r/min for 5min, and measuring the titer of the phage by a double-layer plate method.

As a result, it was found that the optimum multiplicity of infection of the phage was 0.001, under which the titer of the progeny phage produced by infecting the host bacterium with the phage was 1 × 10¹⁰PFU/ml, the phage titer was highest among 8 multiplicity of infection.

(V) propagation and titer determination of phages

Adding 100 mul of host bacteria and phage spot-removing leaching solution into 5ml of 2216E broth, culturing for 3-4 h at 170rpm in an air oscillator at 37 ℃, and obtaining phage proliferation solution after the mixed solution becomes clear. Diluting phage proliferation solution with 2216E broth according to 10 times ratio, mixing 100 μ l sample dilution with 100 μ l Vibrio cholerae uniformly, and preparing double-layer plate with 3 parallel samples for each dilution. During counting, the plaque in the observation plate is counted by taking a plate between 30 and 300, and the titer is calculated.

As a result, the titer of the phage vB-Vchs-PR02 is almost unchanged or slightly reduced within the pH range of 4-11 and still remains at 10⁸The pH value of the phage is wider than PFU/ml.

Example 3 determination of bacteriophage lysis Spectroscopy and in vitro lysis test

(ii) determination of the lysis Profile of bacteriophages

The method for determining the lysis spectrum of the phage by adopting a double-layer plate spreading method comprises the following steps: the bacterial suspension of the host bacteria is obtained according to the method in the example 1, 40 strains of vibrio cholerae separated from hepatopancreas of south America white shrimps in the diseased Shandong Binzhou and Jiangsu Nantong areas are respectively R01-R40, and 40 strains of vibrio cholerae are serotype types of non-O1 and non-O139. And detecting related virulence genes, namely detecting 40 host bacterium enterotoxin genes ctxh, enterotoxin gene ctxB, outer membrane protein gene ompU, zonula junction toxin gene zot, toxin regulatory protein gene toxR, classical biotype (Class-local biotype) toxin co-regulated pilus gene tcpA Class, Eltor biotype (El Tor biotype) toxin co-regulated pilus gene tcpA ET, toxin co-regulated pilus gene tcpI, classical biotype hemolysin gene hlyA Class, Eltor biotype hemolysin gene hlyA ET, heat-stable enterotoxin gene stn/sto and other virulence related genes by PCR.

A cracking spectrum detection method comprises the following steps: respectively adding 100ml of bacterial suspension and 100ml of vB-VchS-PR02 phage into the upper layer agar which is melted and cooled to about 50 ℃, quickly rubbing the test tube by hand to mix uniformly, then quickly pouring the test tube onto the prepared lower layer 2216E nutrient agar plate, slightly shaking to uniformly distribute the test tube, placing the test tube into a 37 ℃ incubator to culture, observing whether phage spots appear or not, and obtaining a lysis spectrum shown in Table 1.

TABLE 1 fragmentation spectra of bacteriophage vB-Vchs-PR02

Note: +: cracking, and brightening plaques; -: it is not cracked.

The experimental results show that: aiming at 40 clinically isolated vibrio cholerae strains, the phage vB-VchS-PR02 can crack 30 vibrio cholerae strains, and the cracking rate of the phage vB-VchS-PR02 reaches 75%.

(II) vB-VchS-PR02 in vitro lysis test (OD value method) for host bacterium R17

Adding host bacteria R17 and bacteriophage vB-Vchs-PR02 according to a certain proportion, wherein the final concentration of the host bacteria is 1 × 10⁸CFU/ml, final concentration of phage 1 × 10⁹PFU/ml，1×10⁸PFU/ml，1×10⁷PFU/ml、1×10⁶PFU/ml, the same amount of sterile 2216E broth as the phage was added to the control, and the mixture of the broth and phage was incubated with shaking at 37 ℃ and 170rpm in a shaker. And measuring OD values at regular intervals until the mixed solution becomes clear, and measuring the residual quantity of each group of bacteria after acting for a certain time by a coating plate method.

The result shows that the vB-Vchs-PR02 has good cracking effect on R17, the cracking efficiency of 4 phages with different concentrations on R17 can reach more than 99 percent, and the best killing effect can be achieved within 4 hours only in different time lengths.

Example 4 kinetics of bacteriophages in the hepatopancreas of Penaeus vannamei

Feeding the dynamics of phage in prawn hepatopancreas by mixing material

Selecting 3g of healthy Penaeus vannamei Boone, taking a water sample in a shrimp tank and the hepatopancreas of the Penaeus vannamei Boone before the test, measuring phages, and feeding 10g of 20 shrimps in each tank mixed with 5 mul 10 when no phages vB-Vchs-PR02 exists⁹PFU/ml vB-Vchs-PR02 feed. The content of the hepatopancreas phage is measured at certain intervals.

The result shows that the hepatopancreas of prawns can detect the phage without the phage vB-Vchs-PR02 before feeding, and the highest titer can reach 10 after the phage vB-Vchs-PR02 is mixed with the feed and fed⁴PFU/ml。

Kinetics in (II) phage water disinfection experiments

The final concentration of the vibrio cholerae in the water body is 1 × 10⁶CFU/ml, adding Vibrio cholerae bacteriophage with MOI of 1 for sterilization.

The result shows that the concentration of the vibrio cholerae in the water body can be reduced to 1 × 10 within 6 hours after the bacteriophage vB-Vchs-PR02 is disinfected³CFU/ml。

(III) the change of mortality rate of bacteriophage in treating vibrio cholerae infection of penaeus vannamei boone

2-3 g of healthy penaeus vannamei boone is selected and divided into 3 groups (blank control group, toxicity attacking group and treatment group), and each group contains 15 shrimps. Taking the hepatopancreas of the same jar of prawns before the experiment, and determining phage in the hepatopancreas by a double-layer plate methodThe titer, when confirming no phage vB-Vchs-PR02 exists, the pathogenic vibrio cholerae 3.5 × 10 is injected into the toxin attacking group and the treatment group⁷Feeding 10g of prawn mixed with 5 μ l of 10 solution 3 hr after toxin counteracting treatment by CFU/prawn⁹PFU/ml vB-Vchs-PR02 feed, the toxicity attacking group was fed with only 10g of normal feed, and the blank control group was not treated. The death of the prawns was observed and recorded.

TABLE 2 control effect of bacteriophage on white feces disease of Penaeus vannamei Boone

The results in table 2 show that the 15-tailed shrimps in the blank control group have no death phenomenon, and the death rate is 0%; the death rate of 14 shrimps in the challenge group is 93%, and the death rate of 8 shrimps in the treatment group is 53%. Compared with the challenge group, the mortality of the treatment group is obviously reduced, which shows that the bacteriophage has a certain control effect on the white feces disease of the penaeus vannamei caused by the vibrio cholerae.

It should be understood that the technical solutions and concepts of the present invention may be equally replaced or changed by those skilled in the art, and all such changes or substitutions should fall within the protection scope of the appended claims.

Claims

1. The novel Vibrio cholerae phage is characterized in that latin is Vibrio cholerae phase, named as vB-Vchs-PR02PR02, and the preservation number is CGMCC NO. 18861.

2. The use of the novel Vibrio cholerae bacteriophage of claim 1 in a medicament or a water disinfectant for preventing and treating Vibrio cholerae infectious diseases.

3. The use according to claim 2, wherein the disease is the white feces syndrome of Penaeus vannamei, and the medicament is administered as a feed additive to Penaeus vannamei.

4. A medicament for the control of diseases caused by infection with Vibrio cholerae, characterized in that the active ingredient comprises the novel Vibrio cholerae bacteriophage of claim 1.

5. The medicament of claim 4, further comprising phages to other specific pathogenic bacteria.

6. An aqueous disinfectant characterized by comprising as an active ingredient the Vibrio cholerae bacteriophage of claim 1; preferably, the concentration of phage is 10⁶PFU/ml or more.

7. The water disinfectant of claim 6, further comprising phages for different species of specific pathogenic bacteria.

8. A detection kit comprising the novel Vibrio cholerae bacteriophage of claim 1.