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Bacteriophage able to split multiple resistant staphylococcus aureus, separation method and application thereof

A staphylococcus, separation method technology, applied in the field of microbiology, can solve the problems of limited number of bacteriophages, narrow bacteriostasis spectrum, and low bacteriostasis efficiency

Inactive Publication Date: 2015-08-19
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the number of phages that have been isolated and utilized so far is very limited, and most of them have poor environmental adaptability, narrow cleavage spectrum, and low cleavage efficiency, especially phages that specifically lyse MRSA strains have rarely been reported in China. , the outstanding advantage of the present invention is to solve the above problems

Method used

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  • Bacteriophage able to split multiple resistant staphylococcus aureus, separation method and application thereof
  • Bacteriophage able to split multiple resistant staphylococcus aureus, separation method and application thereof
  • Bacteriophage able to split multiple resistant staphylococcus aureus, separation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1, the processing of sample

[0043] Fully dissolve the collected soil, feces and nasal swab samples in sterilized 50mL PBS solution, centrifuge at 5000rpm at 4°C for 20min to remove bacteria and other impurities, filter the supernatant with a 0.22μm filter membrane, and place the filtrate in a sterile Shake culture in the bottle at 37°C and 150rpm for 18h, check for sterility, and store the filtrate without bacterial growth at 4°C for enrichment and isolation of phage.

Embodiment 2

[0044] Embodiment 2, enrichment of phage stock solution

[0045] Three enrichment methods were used; the specific operations were as follows:

[0046] After getting the filtrate 30mL of embodiment 1 and the BHI substratum of equal volume and mixing, add and be in logarithmic growth phase (OD 600 =0.5~0.7) of the phage host bacteria Staphylococcus aureus 3mL, shake culture at 165rpm at 37°C overnight. The next day, centrifuge at 12000rpm at 4°C for 15min, take 30mL of the supernatant, add equal volumes of BHI medium and 1mL of host bacteria enrichment solution, stand at 37°C for 30min, shake at 165rpm for 12h, and centrifuge at 12000rpm at 4°C for 15min. Take 15 mL of the supernatant, add an equal volume of BHI medium and 0.6 mL of the host bacteria enrichment solution, let stand at room temperature for 30 min, and shake at 37°C for 4 h at 165 rpm. Centrifuge at 12,000 rpm at 4°C for 20 minutes, collect the supernatant and filter it with a 0.22 μm filter membrane to obtain ...

Embodiment 3

[0047] Embodiment 3, the screening of phage indicator bacteria

[0048] Using 20 clinical isolates of Staphylococcus aureus, 20 MRSA strains, 20 food-borne strains and 17 clinical isolates, a total of 60 strains were used as indicator bacteria, and the double-layer agar plate method was used to screen the suspected plaques. flat. Specific process:

[0049] Take 300 μL of the phage stock solution of Example 2, mix them with 60 strains of the same amount of bacteria cultured overnight, incubate at 37°C for 20 minutes after gently vortexing, add to the upper layer of agar at about 40°C, mix well and quickly pour it on the bottom layer Plate—on a solid plate of BHI medium, place it at room temperature for 15 minutes. After it solidifies, place it upside down in a 37°C incubator and incubate for 11 to 13 hours to obtain a plate that can form phage plaques. Staphylococcus aureus 05L189 was screened as an indicator bacterium.

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Abstract

The invention discloses a bacteriophage able to specifically and efficiently kill multiple resistant staphylococcus aureus, a separation method and application thereof. According to the invention, the bacteriophage able to specifically and efficiently split multiple resistant staphylococcus aureus is screened out from the environment, and the bacteria splitting spectrum is wide. On the basis of a two-layer agar method, a large number of bacteriophages with bacteria splitting activity can be obtained, and the bacteria splitting activity, bacteria splitting spectrum, in vitro bacteria splitting ability and physicochemical properties of the bacteriophages can be determined. The bacteriophage has strong specificity, is difficult to make bacteria generate resistance, also does not produce adverse effect on hosts, and is a feasible method for solving the increasingly serious bacterial resistance problem. The bacteriophage provided by the invention is expected to become a novel antibacterial preparation or environmental disinfectant for preventing and controlling resistant staphylococcus aureus infection.

Description

technical field [0001] The invention belongs to the field of microorganisms, and relates to a phage capable of efficiently and rapidly killing multiple drug-resistant Staphylococcus aureus, and its separation and application. Background technique [0002] Staphylococcus aureus is a common opportunistic pathogen that can cause a variety of serious infectious diseases in humans and animals. The emergence of multidrug-resistant strains of Staphylococcus aureus has made it increasingly difficult to treat infections caused by this bacteria. In particular, the emergence of Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Staphylococcus aureus (VRSA) strains has shown a strong response to almost all commonly used antibiotics. Extremely drug resistant. Under such circumstances, even if a large amount of manpower and funds are invested to continue to develop new antibiotics, the problem of bacterial drug resistance cannot be effectively solved. Therefore...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N7/02A61K35/76A61P31/04A01N63/02A01P1/00
Inventor 严亚贤郑盼盼孙建和
Owner SHANGHAI JIAO TONG UNIV
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