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Edwardsiella fish killing bacteriophage, and bacteriophage composition and application thereof

A phage and composition technology, applied in the field of microbiology, can solve the problem of not finding specific cracking and killing Edwardsiella pisces, achieve high acid-base stability and thermal stability, safe use, and reduce the incidence of disease

Active Publication Date: 2022-07-12
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, no phage resources that specifically lyse Edwardsiella piscida have been found so far, and the existing technology needs to be further developed

Method used

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  • Edwardsiella fish killing bacteriophage, and bacteriophage composition and application thereof
  • Edwardsiella fish killing bacteriophage, and bacteriophage composition and application thereof
  • Edwardsiella fish killing bacteriophage, and bacteriophage composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Isolation and purification of phage

[0050] (1) Preparation of host bacterial suspension

[0051] Take Edwardsiella fishicidal RE03 and streak it on the TSA plate, pick a single colony and inoculate it into 5 mL of TSB liquid medium, and proliferate to the logarithmic growth phase under the conditions of 170 rpm and 28 °C to obtain a fresh bacterial solution.

[0052] (2) Isolation and purification of bacteriophage

[0053] (1) Isolation of phage

[0054] The sewage collected from a seafood market in Qingdao City, Shandong Province was centrifuged at 11,000 r / min for 5 minutes. After the supernatant obtained by centrifugation was filtered with a 0.22 μm filter membrane, TSB medium and 100 μL of host bacteria suspension were added, and the solution was heated at 170 rpm. After culturing at 28°C for 48h, centrifuge at 11000r / min for 5min and filter with a 0.22μm membrane to obtain the primary enrichment phage stock solution; add the primary enrichment phage s...

Embodiment 2

[0063] Example 2 Identification of phage

[0064] Electron microscopy of bacteriophages

[0065] 1. Experimental method:

[0066] Take 20 μl for a titer of 10 9 PFU / mL of the previously analyzed phages were dropped on the copper mesh, precipitated for 15 min, the excess liquid was absorbed with filter paper, stained with 2% phosphotungstic acid for 30 min, and observed by electron microscope after drying.

[0067] 2. Experimental results and analysis:

[0068] The result is as figure 1 As shown in the figure, the length of the head of the phage is 58 nm, the width of the head is 63 nm, and the length of the tail is about 8 nm. According to the classification standard of the ninth report of the International Committee on Taxonomy of Viruses (ICTV), the phage can be identified. It belongs to the family Brachyphage and was named PRE03.

Embodiment 3

[0069] Example 3 Determination of biological characteristics of bacteriophage

[0070] (1) Determination of lysis profile of bacteriophage

[0071] 1. Experimental method:

[0072] (1) Experimental object: The lysed object is 85 fish-killing Edwardsiella strains, and these 85 strains are derived from flounder, sea bream and sea bream from Dalian, Liaoning, Yantai, Shandong, Rizhao, Shandong, Weihai, Shandong, Qingdao, Fujian, and Zhanjiang, Guangdong. Sea bass and water samples.

[0073] (2) PCR virulence gene detection was performed on these 85 strains to detect whether they carried pilin precursor (fimA), killing factor (mnkF), catalase precursor (katB), and glutamate decarboxylase. Isozyme (gadB) and citrate lyase (citC) five virulence genes.

[0074] (3) In this example, the double-layer plate method was used to determine the phage lysis spectrum. 100 μL of the phage suspension and the Edwardsiella fish-killing bacteria solution were added to a 0.5 mL centrifuge tube an...

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Abstract

The invention discloses an Edwardsiella fish killing bacteriophage, a bacteriophage composition and application thereof, the bacteriophage is named as PRE03, and is preserved in China General Microbiological Culture Collection Center on January 7, 2022, the preservation address is No.3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.45074. The invention further discloses a preparation method of the Edwardsiella fish killing bacteriophage, the Edwardsiella fish killing bacteriophage, the Edwardsiella fish killing bacteriophage composition and the application of the Edwardsiella fish killing bacteriophage composition. The invention further provides a bacteriophage composition, and the bacteriophage composition comprises the Edwardsiella fish killing bacteriophage PRE03. The bacteriophage not only has strong infection ability and lysis ability, but also has high thermal stability and acid-base stability, is a bacteriophage capable of efficiently killing Edwardsiella fish, and is safe to use and wide in application range; the bacteriophage and the bacteriophage composition thereof can be effectively used for preventing and treating infection caused by Edwardsiella fish killing, and can be widely applied to various links which are easy to cause loss due to infection of Edwardsiella fish killing in an aquaculture process, daily disinfection of an aquaculture environment, bacteriostasis of fresh aquatic foods and the like.

Description

technical field [0001] The present invention relates to the technical field of microorganisms, in particular to a fish-killing Edwardsiella phage, its phage composition and its application. Background technique [0002] Edwardsiella piscicida belongs to the family Enterobacteriaceae and the genus Edwardsiella, and is a Gram-negative bacterium, short rod-shaped and motile. The host range of Edwardsiella ichthyosa is mostly marine and freshwater fish, including barramundi, European eel, sea bream, turbot, largemouth bass and black-spotted stingray. Fish-killing Edwardsiella can cause Edwardsiosis, a widespread disease that can cause extensive skin lesions, necrotizing abdominal swelling, visceral cysts, and hemorrhagic sepsis after infection in fish. The outbreak of Edwardsiosis pathogenic bacteria has brought serious social and economic losses to countries in the United States, Europe, and Asia (including Japan, China, etc.). [0003] At present, the existing methods mainly...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/04A61K35/76A61P31/04A23K10/18A23K50/80A01N63/40A01P1/00C12R1/92C12R1/01
CPCC12N7/00C12Q1/04A61K35/76A61P31/04A23K10/18A23K50/80A01N63/40C12N2795/10221C12N2795/10232C12N2795/10231G01N2333/24Y02A40/81
Inventor 潘强任慧英孙虎芝谢金红侯文秋马雪妮徐帆
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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