CN111110671A

CN111110671A - Application of fraxins in resisting aquatic animal streptococcus agalactiae infection

Info

Publication number: CN111110671A
Application number: CN202010057072.5A
Authority: CN
Inventors: 董靖; 艾晓辉; 刘永涛; 胥宁; 杨秋红; 杨移斌; 周顺
Original assignee: Yangtze River Fisheries Research Institute CAFS
Current assignee: Yangtze River Fisheries Research Institute CAFS
Priority date: 2020-01-17
Filing date: 2020-01-17
Publication date: 2020-05-08

Abstract

The invention discloses an application of fraxins in resisting aquatic animal streptococcus agalactiae infection, wherein the fraxins can reduce the adhesion effect of streptococcus agalactiae by inhibiting the activity of sortase A, so as to reduce the pathogenicity of streptococcus agalactiae to aquatic animals.

Description

Application of fraxins in resisting aquatic animal streptococcus agalactiae infection

Technical Field

The invention belongs to the technical field of aquaculture, and particularly relates to application of fraxins in preparation of a medicament for preventing or treating streptococcus agalactiae infection of aquatic animals, or an aquatic feed additive for resisting streptococcus agalactiae infection.

Background

The ash trees are also called areca-nut and white wattle, belong to deciduous arbors of the family of Oleaceae, have a long cultivation history and are widely distributed in China, and the bark of the ash tree is mostly used as a medicine. The white wax bark is mild in nature, pungent in taste and non-toxic; it enters liver, gallbladder and large intestine meridians. Is mainly used for treating malaria, menstrual disorder and infantile head sore. Modern medical research shows that the fraxinus excelsior has the functions of diminishing inflammation, easing pain, influencing urine volume, uric acid excretion and the like. It is clinically used for treating bacillary dysentery, chronic tracheitis, etc. and has good effect.

Streptococcus agalactiae is a common opportunistic pathogen, belongs to group B streptococcus, is distributed all over the world, can cause various infections of terrestrial animals, fishes and people, and is a typical human-animal-fish co-infected pathogen. In aquaculture, streptococcus agalactiae can infect a plurality of cultured fishes, is one of main pathogenic bacteria of tilapia, and brings serious threat to the healthy development of freshwater aquaculture industry in China. In addition, since streptococcus agalactiae can cause various infections of terrestrial animals and humans, and consumption of water products has been increasing year by year in recent years, the infection of streptococcus agalactiae has attracted more and more attention, and streptococcus agalactiae is one of the hidden troubles affecting food safety and public safety. Therefore, enhancing control of streptococcus agalactiae in an aquaculture environment is of great importance to the healthy farming and human health of tilapia. Chemical antibacterial drugs are the main means for treating bacterial infection of cultured animals in aquaculture, but the treatment of bacterial diseases is increasingly difficult due to the occurrence of pathogenic bacteria drug resistance and even the generation of multiple drug-resistant bacteria caused by the unreasonable use of antibacterial drugs. In addition, when the drug resistance gene of the aquatic-derived drug-resistant bacterium is exchanged or transmitted with the human-derived pathogenic bacterium, the drug resistance of the human-derived pathogenic bacterium is generated or increased. The use of a large amount of antibacterial drugs not only causes serious drug resistance, but also causes the residue of chemical antibacterial drugs in fish bodies by blindly increasing the dosage, thereby causing residue and risk and potential influence on the quality of aquatic products. Therefore, enhanced control of pathogenic bacteria of aquatic origin is of great importance for aquaculture, human health and food safety. The tilapia streptococcus agalactiae is mainly treated by chemical antibacterial drugs, so that the treatment effect is poor, the generation of drug-resistant bacteria is caused, and the healthy culture of tilapia is seriously damaged. Therefore, the development of new mechanism drugs to replace chemical antibacterial drugs in aquaculture is urgently needed.

Sortase is a key enzyme of gram-positive bacteria to anchor surface pathogenic proteins to cell walls, and is a new target for drug development against gram-positive bacteria. Sortase a mediated surface proteins are mainly associated with adhesion and invasion of streptococcus agalactiae, which are the first steps of infection. Therefore, the development of drug screening with the sortase A as a target has wide prospect and important significance for controlling the streptococcus agalactiae infection of the aquaculture animals.

Disclosure of Invention

The invention aims to provide application of the fraxins in resisting infection of aquatic animals by streptococcus agalactiae, wherein the fraxins can inhibit the activity of streptococcus agalactiae sortase A and further reduce the adhesion of streptococcus agalactiae to aquatic animals so as to reduce the pathogenicity of the streptococcus agalactiae.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of the fraxins in resisting the infection of aquatic animals with streptococcus agalactiae comprises the following steps: experiments prove that when the concentration of the aesculin reaches more than 8 mug/mL, the catalytic activity of the sortase A is remarkably inhibited; when the concentration of the fraxins reaches more than 8 mug/mL, the adhesion of streptococcus agalactiae to fibrinogen and fibronectin can be remarkably inhibited; the tilapia infected with streptococcus agalactiae is drenched with the fraxins with the dose of 50mg/kg once every 12h and is continuously used for 3 days, and the death rate of the tilapia in the 5 th day is reduced from 100 percent of a control group to 40 percent, so that the fraxins have a better treatment effect on the streptococcus agalactiae.

The fraxins are the main chemical components of the bark of Chinese medicinal Fraxinus chinensis, have no toxic or side effect and no residue, and can be used as a medicinal preparation and a feed additive for preventing and treating the streptococcus agalactiae of aquatic animals.

Compared with the prior art, the invention has the following advantages and beneficial effects: the traditional Chinese medicine composition has the advantages of low dosage, good treatment effect and difficult generation of drug resistance of pathogenic bacteria, has no toxic effect on fish bodies, people and the environment because the main components are natural compounds derived from traditional Chinese medicines, and has no risk of drug residue.

Drawings

FIG. 1 shows the effect of fraxidin on catalytic activity of sortase A

FIG. 2 is a graph showing the effect of fraxidin on adhesion of Streptococcus agalactiae

Detailed Description

Example 1: determination of minimum inhibitory concentration of aesculin to streptococcus agalactiae

The Streptococcus agalactiae strain PBSA0903 related by the invention is given by doctor Guoweilang of Hainan university (Monee cloud, Zhouyanlaung, Huwenting, Wumeng dao, Wangsheng, Xizhenyuyu, Guoweirang. the influence of temperature on hemolytic valence of 14 Streptococcus agalactiae strains [ J ]. proceedings of Hainan university (Nature science edition), 2016,34(02): 170-.

The minimum inhibitory concentration of fraxidin against PBSA0903 was determined using the broth microdilution method recommended by CLSI. The method comprises the following steps:

(1) diluting prepared aesculin (purchased from Shanghai leaf Biotechnology Co., Ltd., product number: B20990) in 96-well cell culture plate in a multiple ratio, wherein the concentration is 512 mu g/mL, 256 mu g/mL, 128 mu g/mL, 64 mu g/mL, 32 mu g/mL, 16 mu g/mL, 8 mu g/mL, 4 mu g/mL, 2 mu g/mL and 1 mu g/mL respectively; the volume of the medicine in each hole is 100 mu L;

(2) respectively inoculating single colony of PBSA0903 strain to THB culture medium at 37 deg.C, culturing overnight, centrifuging, collecting thallus, adjusting concentration to 0.5 McLeod turbidity with sterile physiological saline, and diluting to 1 × 10 with culture medium⁶CFU/mL, the final concentration of 5X 10 was achieved by adding the bacterial suspension to a 96-well cell culture plate⁵CFU/mL, respectively setting negative and positive control groups (negative control group without drug and bacterial solution, positive control group with bacterial solution and no drug), repeating three times for each drugAnd secondly, observing the result after culturing for 18-24h in a biochemical incubator at 30 ℃, and judging the lowest inhibitory concentration of the drug according to the lowest drug concentration without bacterial growth. As a result, the minimum inhibitory concentration of the aesculin on the Streptococcus agalactiae PBSA strain is more than 512 mug/mL.

Example 2: method for determining inhibition effect of aesculin on streptococcus agalactiae sortase A by energy transfer resonance method

The sortase A (reference: Khare, B., et al, preliminarysterigmatographic study of the Streptococcus agalactiae startases, sortase A and dsortase C1.acta Crystallogr Sect F Struct Biol Crystal Commun,2010.66(Pt 9): p.1096-100.) related by the present invention is preserved by aquatic animal pharmacology and drug residue control technology laboratory of Yangtze river aquatic research institute, and the Streptococcus agalactiae PBSA0903 is preserved by aquatic research institute of Yangtze river. The specific test steps are as follows: 3. mu.L of 6mg/mL stock solution of sortase A protein, 10. mu.L of 200. mu.M stock solution of fluorogenic substrate (Dabcyl-QALPETGEE-Edans) were added to the black opaque 96-well plate, and then the stock solution of aesculin (purchased from Shanghai-derived leaf Biotech Co., Ltd., product number: B20990) at a concentration of 10240. mu.g/mL was added to make the concentration of 32, 16, 8, 4, 2, 0. mu.g/mL, and buffer solution (50mM Tris-HCl,150mM NaCl,5mM CaCl) was added to each well₂pH 7.5)) the reaction system was 200 μ L, 3 replicate wells per drug concentration. Put into a 37 ℃ incubator in a dark place to be incubated for 1h, then taken out, and the change of the fluorescence value is measured by using an enzyme-linked immunosorbent assay. The excitation wavelength was 350nm and the recording wavelength was 490 nm. The influence of different concentrations of aesculin on the catalytic activity of the SrtA protein was analyzed by fluorescence value change. As shown in FIG. 1, the catalytic activity of sortase A was significantly inhibited (0.01 < p < 0.05) when the concentration of fraxins reached 8. mu.g/mL, and was significantly inhibited (p < 0.01) when the concentration reached 16. mu.g/mL or more.

Example 3: effect of Fraxins on the adhesive function of Streptococcus agalactiae mediated by sortase A

Fibrinogen and fibronectin stock solutions were diluted to 4. mu.g/mL with PBS, 200. mu.L was added to a 96-well cell culture plate, and incubated overnight at 4 ℃. The following day the plate was drained and washed 3 times with 200. mu.L PBS, 5% per wellBSA was blocked in a 37 ℃ incubator for 2 h. The liquid was then discarded and washed 3 times with PBS until use. Selecting single colony of Streptococcus agalactiae PBSA0903 strain, inoculating to 2mL BHI culture medium, culturing at 37 deg.C for 8 hr, expanding to 100mL culture medium, adding 32, 16, 8, 4, 2, and 0 μ g/mL aesculin, and culturing to OD_600nmThe cells were collected by centrifugation at 0.5 ℃. The cells were washed with PBS 3 times and then adjusted to OD_600nmIs 1.0 for standby. To the closed 96-well cell culture plate, 100. mu.L of the treated bacterial suspension was added, and the mixture was incubated at 37 ℃ for 2 hours in an incubator. Discarding PBS from the plate, washing for 3 times, oven drying at 50 deg.C, adding 200 μ L of 0.4% crystal violet, dyeing for 20min, washing for 3-5 times, adding 100 μ L glacial acetic acid, dissolving crystal violet thoroughly, and determining OD_570nmThe absorption value of (b). As shown in FIG. 2, the adhesion of Streptococcus agalactiae PBSA0903 to fibrinogen and fibronectin is reduced sequentially with the increase of the drug concentration, and when the concentration of the fraxins reaches more than 8 μ g/mL, the fraxins can remarkably inhibit the adhesion of Streptococcus agalactiae PBSA0903 to fibrinogen and fibronectin.

Example 4: therapeutic effect of fraxin on tilapia streptococcus agalactiae infection model

The test was carried out in aquaria of 100cm by 50cm by 60cm, with water changes and water temperature maintained at 30. + -. 2 ℃ per day. After being anesthetized, about 100g of healthy tilapia is injected with streptococcus agalactiae PBSA0903 strain to establish an artificial infection model. And (3) irrigating 50mg/kg of fraxin to the infected tilapia 2h after infection, continuously administering for 3d once every 12h, and irrigating PBS as a control after the positive control group is infected. 20 tilapia tails were used in each experimental group and death was recorded immediately after infection, and the results are shown in Table 1. The result shows that the tilapia completely dies within 5 days after being artificially infected with streptococcus agalactiae; 40% of deaths occurred in tilapia treated with 50mg/kg fraxins; the survival rate is far higher than that of the positive control group. The research fully shows that after tilapia is infected with streptococcus agalactiae, the tilapia can be treated by administering the fraxinin with the dose of 50mg/kg, and the medicament can obtain better treatment effect.

TABLE 1 mortality rate following treatment of Ormosia tilapia Streptococcus agalactiae infection model with Fraxins

It was found by example 1 that fraxins had no inhibitory effect on the growth of tilapia-derived streptococcus agalactiae in vitro. It was found by example 2 that the aesculin dose-dependently reduced the catalytic activity of the streptococcus agalactiae sortase a in vitro. The sortase A is an important virulence factor of the streptococcus agalactiae, is a new target for researching streptococcus agalactiae medicaments, and is closely related to adhesion, invasion, biofilm formation and the like of the streptococcus agalactiae. Thus, the study of example 3 was further conducted, and as a result, it was found that the aesculin can reduce the adhesion of Streptococcus agalactiae to human fibrinogen and fibronectin. The above studies indicate that fraxins can reduce adhesion in vitro by inhibiting the activity of streptococcus agalactiae sortase a. Adherence and invasion are the first steps of bacterial invasion into the host, and are also critical steps. Therefore, the treatment effect of the fraxin on a tilapia streptococcus agalactiae infection model is further researched, and the result of example 4 shows that the survival rate of infected tilapia is remarkably improved compared with a positive control group after the treatment of the fraxin with the dose of 50mg/kg, and the result shows that the fraxin has a better treatment effect on tilapia streptococcus agalactiae infection and can be used as a medicament for resisting the tilapia streptococcus agalactiae infection.

Streptococcus agalactiae is widely present in aquaculture environment, can infect various warm water fishes such as tilapia, channel catfish, turbot, genuine porgy and schizothorax prenanti, and sortase A is a transpeptidase contained in gram-positive bacteria including Streptococcus agalactiae, and has universality. Therefore, the fact that the fraxins have treatment effect on the streptococcus agalactiae infection of the aquaculture animals can be inferred.

Claims

1. The application of the fraxins as aquatic feed additives in resisting streptococcus agalactiae infection.

2. Application of cortex Fraxini in preparing medicine for preventing or treating Streptococcus agalactiae infection of aquatic animals is provided.

3. Use according to claim 2, characterized in that the dose of fraxidin administered is greater than 50 mg/kg.