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Citrobacter freundii bacteriophage, bacteriophage composition and application thereof

A technology of citric acid freundii and bacteriophage, applied in the field of microorganisms, can solve problems such as phage resources that have not been found in Citrobacter freundii disease, and achieve the diversification of application methods, reduce morbidity and mortality, and reduce the probability of disease incidence. Effect

Pending Publication Date: 2022-07-19
QINGDAO PHAGEPHARM BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are no phage resources available for the prevention and treatment of Citrobacter freundii

Method used

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  • Citrobacter freundii bacteriophage, bacteriophage composition and application thereof
  • Citrobacter freundii bacteriophage, bacteriophage composition and application thereof
  • Citrobacter freundii bacteriophage, bacteriophage composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Isolation and culture of bacteriophage

[0044] (1) Preparation of host bacterial suspension

[0045] Citrobacter freundii RF03 was streaked on a TSA plate in three areas, and cultured upside down at 37°C for 16 hours to obtain a uniform single colony. The single colony was picked and inoculated into 5 mL of TSB liquid medium, at 170 rpm, 37 Proliferate to logarithmic growth phase under the condition of ℃ to obtain fresh host bacteria liquid.

[0046] (2) Isolation and purification of bacteriophage

[0047] 1. Phage isolation

[0048] The sludge collected from a bay area in Haikou City, Hainan Province was added to the bubble sample bottle, and about 150 mL of double-distilled water was added to mix well, 200 μL of bacterial solution was added and 50 mL of TSB solution was added to mix well, and enriched at 37°C for 24 hours; the enrichment was over. Then, centrifuge at 11000r / min for 10min, filter the supernatant through a 0.22um filter to obtain a phage s...

Embodiment 2

[0058] Example 2 Determination of biological characteristics of bacteriophage

[0059] (1) Electron microscope detection of bacteriophages

[0060] 1. Experimental method:

[0061] Take 20 μL of titer of 10 9 PFU / mL of the phage to be tested was 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.

[0062] 2. Experimental results and analysis

[0063] like figure 1 The phage has a head length of 73 nm, a diameter of about 68 nm, a tail length of about 63 nm, and a retractable tail sheath, according to the classification of the ninth report of the International Committee on Taxonomy of Viruses (ICTV). Standard identification, it can be determined that the phage belongs to the Myotail phage family, and it is named PRF01.

[0064] (2) Determination of lysis profile of bacteriophage

[0065] 1. Host bacteria:

[0066] The host ...

Embodiment 3

[0110] Example 3 The effect of phage PRF01 injection on the metabolic process of Crayfish in vivo

[0111] 1. Experimental method

[0112] Healthy Crayfish with a weight of about 20 g were selected and fasted overnight. One control group and three experimental groups were set up, with 40 in each group. Do the following to it:

[0113] The shrimps in the control group were injected with 0.1 mL of TSB medium, and the injection was carried out through the body cavity injection method, which was injected from the base of the second appendage of Crayfish. The phage content is 1×10 8 PFU / mL. After injection, the contents of phage in the shrimp intestine, hepatopancreas of the tested shrimp and the normal shrimp in the blank control group were detected at 0.5h, 5h, 10h, 24h, 48h, 72h, 96h, 120h, and 144h, respectively.

[0114] 2. Experimental results and analysis

[0115] 30 minutes after the injection of phage PRF01, the existence of phage was detected in the shrimp intestine ...

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Abstract

The invention discloses a citrobacter freundii bacteriophage, a bacteriophage composition thereof and application of the citrobacter freundii bacteriophage, the citrobacter freundii bacteriophage is named as PRF01, the citrobacter freundii bacteriophage 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. 45075. The invention also provides a bacteriophage composition which comprises the citrobacter freundii bacteriophage. The citrobacter freundii bacteriophage and the bacteriophage composition can be applied to preparation of drugs, aquatic feed additives or environmental disinfectants for preventing and treating diseases caused by citrobacter freundii infection. The bacteriophage has excellent splitting performance on citrobacter freundii, is wide in splitting spectrum, wide in application range and free of side effects, and can effectively prevent and treat infection caused by the citrobacter freundii and various water body problems caused by mass proliferation of pathogenic bacteria in water bodies.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a phage of Citrobacter freundii, its phage composition and its application. Background technique [0002] Citrobacter freundii is a gram-negative short bacterium belonging to the genus Citrobacter Werkman and Gillen of the family Enterobacteriaceae. Opportunistic pathogens of fish comorbidities. In aquatic animals, Citrobacter freundii was first isolated from the sunfish (Mola mola). [0003] In recent years, Citrobacter freundii has affected the health of many aquatic animals due to its strong ability to proliferate and infect. Studies have shown that Citrobacter freundii easily infects Crayfish, causing sepsis. At the same time, many studies have confirmed that Citrobacter freundii can cause disease in fish. There have been reports of Citrobacter freundii causing the disease of Atlantic salmon, rainbow trout, salmon, koi, zebrafish, sturgeon, etc. The diseased fish ar...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12Q1/04A61K35/76A61P31/04A23K10/18A23B4/22A01N63/40A01P1/00C12R1/92C12R1/01
CPCC12N7/00C12Q1/04A61K35/76A61P31/04A23K10/18A23B4/22A01N63/40C12N2795/10121C12N2795/10131C12N2795/10132G01N2333/24Y02A40/81
Inventor 潘强任慧英孙虎芝侯文秋谢金红马雪妮徐帆
Owner QINGDAO PHAGEPHARM BIO TECH CO LTD
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