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Staphylococcus aureus bacteriophage LSA2311 and application thereof

A Staphylococcus, golden yellow technology, applied in the direction of phage, virus/phage, application, etc., can solve the problem of narrow host spectrum and achieve the effect of wide host spectrum, high titer, and good lysis effect

Active Publication Date: 2021-02-02
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The limitation of the application of phage therapy is the narrow host spectrum

Method used

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  • Staphylococcus aureus bacteriophage LSA2311 and application thereof
  • Staphylococcus aureus bacteriophage LSA2311 and application thereof
  • Staphylococcus aureus bacteriophage LSA2311 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The isolation and screening method of staphylococcus aureus phage LSA2311, its step is:

[0043] (1) Sample collection

[0044] The sewage samples came from a vegetable market in Wuhan City, Hubei Province.

[0045] (2) Screening of Staphylococcus aureus phage

[0046] Take 10 mL of sewage sample and filter it with a 0.22 μm microporous filter. Put into 20mL sterilized LB broth medium (the composition of the medium is: tryptone 10.0g / L, yeast extract 5.0g / L, sodium chloride 10.0g / L, pH value 7.3±0.2 The method of using the culture medium is as follows: take 25.0 g of the LB broth culture medium of the above-mentioned ingredients, dissolve it in 1000 mL of distilled water with heating and stirring, distribute it in test tubes or other suitable containers, and autoclave at 121 ° C for 20 min for later use) Into a 50mL sterilized centrifuge tube, add 5mL of the host bacteria solution in the logarithmic growth phase (6h-8h of culture). Shake culture at 37°C for 12h-18h ...

Embodiment 2

[0056] Example 2: Determination of bacteriophage LSA2311 host spectrum

[0057] 29 strains of Staphylococcus aureus (ATCC25923, ATCC29213, 6538, 196, 7, 16, 18, 30, 32, SA2, SA6, SA24, SA26, SA90, SA99, SA100, SA102, SA103, SA104, SA105, SA106、SA109、SA113、SA115、SA118、SA122)和9种其他菌属(鼠伤寒沙门氏菌SJTUF13277、SJTUF13350、SJTUF13336、ATCC10855、SJTUF13337、SJTUF13277、肠炎沙门氏菌10960、印第安纳沙门氏菌13500、大肠杆菌ATCC25922)来做噬菌体LSA2311 The host spectrum analysis, the specific steps are as follows:

[0058] The above-mentioned strains (29 strains of Staphylococcus aureus and 9 strains of other species) were cultured to logarithmic phase, respectively. Take 100 μL of the above bacterial solution in the logarithmic phase, and when the temperature of the upper layer of agar (add 0.7 g of agar to 100 mL of the LB broth medium in step (2) of Example 1, and sterilize at 121 ° C for 20 min for later use), when the temperature drops to 40 ° C, respectively Take 3mL of the upper layer of agar and mix it with the abov...

Embodiment 3

[0065] Embodiment 3: Electron microscope observation of bacteriophage LSA2311

[0066] Using phosphotungstic acid negative staining method (Clokie and Kropinski 2009), the phage suspension was ultracentrifuged at 4°C and 40,000r / min for 1h to precipitate the phage particles, and the pellet was resuspended with 0.1mol / L ammonium acetate. Take 20 μL of the phage suspension and volume 20 μL of phosphotungstic acid with a fraction of 2% and pH=7 was added dropwise on the parafilm respectively. Take the copper grid lightly, first immerse it in the phage liquid for 10 minutes, and then absorb the excess liquid with filter paper. Then place the copper grid in phosphotungstic acid dye for 10 minutes, absorb the excess liquid, and let it dry naturally until it is completely dry. Observe the shape of the phage under the transmission electron microscope on the prepared copper grid, and measure its size with the software DigitalMicrograph Demo 3.9.1 .

[0067] The result is as figure ...

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Abstract

The invention discloses a staphylococcus aureus bacteriophage LSA2311 and application thereof. The staphylococcus aureus bacteriophage LSA2311 is of a wide spectrum type and can carry out lysis on staphylococcus aureus and drug-resistant strains thereof. An authentication result shows that the bacteriophage is of caudovirales myoviridae, the collection number of the bacteriophage is CCTCC NO: M 2020562, and the bacteriophage LSA2311 has a stable titer when pH is 6-11 and temperature is 40-50 DEG C. A complete genome sequencing result of the bacteriophage disclosed by the invention shows that the bacteriophage does not have virulence or antibiotic resistance and can transduce related genes, and safety generated when the bacteriophage disclosed by the invention is applied to treatment of pathogenic bacteria and bacteriophage in food can be proved from a genetic background. By use of the bacteriophage provided by the invention, the staphylococcus aureus in a milk sample can be effectivelycontrolled. Compared with antibiotics and chemical preservatives, the bacteriophage provided by the invention has the characteristics of being high in specificity, free from residues and safe.

Description

technical field [0001] The invention relates to the field of food safety, in particular to a Staphylococcus aureus phage LSA2311 and an application thereof. Background technique [0002] As one of the most common foodborne pathogens, Staphylococcus aureus (S.aureus) is closely related to the hygiene and safety of the food industry and the medical field. Because it widely exists in nature, it is distributed in air, soil, water and tableware, and both humans and animals have a high carrier rate. It is reported that the carrier rate of Staphylococcus aureus in the normal population can reach 30%-80% %. As one of the important sources of foodborne diseases, Staphylococcus aureus has caused more and more food poisoning incidents, which has greatly affected the field of food hygiene. According to reports, there are about 185,060 food safety incidents caused by Staphylococcus aureus enterotoxin in the United States every year, of which about 1,750 people are hospitalized, account...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00A01N63/40A01P1/00A23C9/12A23C3/00C12R1/92
CPCC12N7/00A01N63/40A23C9/1203A23C3/00C12N2795/10121C12N2795/10131
Inventor 王小红陈慕潇王佳丁一峰朱文娟张宇
Owner HUAZHONG AGRI UNIV
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