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Pseudomonas aeruginosa phage and application thereof

A technology of Pseudomonas aeruginosa and phage, applied in the direction of phage, virus/phage, application, etc., can solve the problems of general effect, achieve good effect, obvious inhibitory effect, and enhanced scavenging effect

Active Publication Date: 2017-07-07
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the international research on the inhibition of Pseudomonas aeruginosa BF is mainly devoted to the research of antibiotics. At present, it has been found that fourteen- and fifteen-membered ring macrolide antibiotics (such as erythromycin, clarithromycin, azithromycin), and rifampin are Can inhibit the formation of BF by Pseudomonas aeruginosa in vitro, but the effect is general

Method used

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  • Pseudomonas aeruginosa phage and application thereof
  • Pseudomonas aeruginosa phage and application thereof
  • Pseudomonas aeruginosa phage and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Isolation and Identification of Phage

[0036] Isolation of a phage

[0037] Take 1L of sewage before hospital treatment, add CaCl 2 Centrifuge at 5000rpm for 10min at 4°C to 1mmol / L, remove the precipitated particles in the sewage, take the supernatant and filter it with a 0.22μm filter membrane to sterilize; take 20mL of the filtrate and mix it with 20mL 2×LB medium, and use 1% inoculum , inoculate 400 μL of Pseudomonas aeruginosa, enrich and culture at 37°C for 12h. Take 5ml of the above bacteria solution and centrifuge at 4°C and 5000rpm for 10min, take the supernatant and filter it with a 0.22μm filter membrane to obtain the phage stock solution. Dilute the obtained phage stock solution 10 times in a gradient, take 300 μL of the diluted solution and mix it with the corresponding Pseudomonas aeruginosa in logarithmic growth phase 1:1, incubate at 37°C for 15min, mix with 4mL of 55°C liquid LB medium, and pour evenly into On a solid LB agar plate, cool f...

Embodiment 2

[0050] Example 2 Pseudomonas aeruginosa phage (CGMCC No.13381) inhibits the detection of Pseudomonas aeruginosa biofilm

[0051] 1 Amplification and purification of phage (CGMCC No.13381)

[0052] Pick a single colony of host bacteria, inoculate it in 100mL liquid LB medium, culture at 37°C, 140rpm for 6 hours to the early logarithmic growth stage, then pick the above-mentioned purified phage plaques with a sterilized pipette tip and add them to the logarithmic growth stage In the host bacteria, culture at 37°C, 140rpm for about 6h, when white flocs are observed; take out the culture at 8000rpm, centrifuge for 5min, take 10ml of the supernatant and add 50ml of the host strains cultivated to the logarithmic growth phase, at 37°C, Cultivate at 140rpm, observe the state of the culture medium, after the culture medium becomes relatively clear and white flocculent precipitates appear (about 5-7h), centrifuge the culture medium at 4°C and 8000rpm for 5min, take the supernatant and u...

Embodiment 3

[0059] Example 3 Detection of Pseudomonas aeruginosa bacteriophage CGMCC No.13381 destroying Pseudomonas aeruginosa biofilm

[0060] 1 Amplification and purification of phage (CGMCC No.13381)

[0061] As described in Example 2

[0062] 2 Activation of Pseudomonas aeruginosa

[0063] As described in Example 2

[0064] 3 The cultivation of biofilm and the effect of phage on it

[0065] Will be cultivated to the logarithmic growth phase (about 10 8 Pfu / mL) of Pseudomonas aeruginosa was inoculated into a 24-well cell culture plate containing cell slides, 20 μL of bacterial solution and 2 mL of TSB medium in each well, and cultured in a 37°C incubator for 24 hours, and the medium was replaced once , adding 2 mL of TSB medium to continue culturing for 48 hours. After culturing for 72 hours, the cell slides were taken out, gently washed twice with sterilized PBS (pH7.2) to remove free bacteria, and then the cell slides were replaced into a new 24-well plate to add phage (MOI=0.1...

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Abstract

The invention provides pseudomonas aeruginosa phage and application thereof. According to the pseudomonas aeruginosa phage, the strain name is vB_PaeM_QKL1, the preservation number is CGMCC No.13381, the classification name is pseudomonas aeruginosa phage, and the pseudomonas aeruginosa phage was preserved in China General Microbiological Culture Collection Center (CGMCC) in December 8th, 2016. The invention also provides application of the pseudomonas aeruginosa phage. Degrading enzyme of an extracellular polymeric substance exists in the caudal spine of the pseudomonas aeruginosa phage and can crack cell envelope to crack biofilm generating bacteria so as to completely remove the bacterial biofilm; the pseudomonas aeruginosa phage provided by the invention has remarkable inhibition effect on a pseudomonas aeruginosa biofilm, can break the pseudomonas aeruginosa biofilm, and also can be combined with antibiotics and the common chemical detergents.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a Pseudomonas aeruginosa phage and its application. Background technique [0002] Pseudomonas aeruginosa (P.Aeruginosa), also known as Pseudomonas aeruginosa, is a Gram-negative pathogenic bacterium that exists widely in the environment and can cause cystic fibrosis in humans, as well as severe burns and endangering certain cancer patients. Acute infection in life and chronic infection in immunosuppressed patients. In animals, Pseudomonas aeruginosa mainly causes endometritis in horses, mastitis in cattle and other ruminants, otitis media and otitis interna in chinchillas, hemorrhagic pneumonia in minks, etc., which has brought huge damage to the breeding industry. loss. The traditional method of treating Pseudomonas aeruginosa infection is antibiotics. However, due to the emergence of multidrug-resistant Pseudomonas aeruginosa strains and the shortage of new therap...

Claims

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Application Information

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IPC IPC(8): C12N7/00A01N63/00A01P1/00
CPCA01N63/00C12N7/00C12N2795/10121
Inventor 徐永平渠坤丽王丽丽李晓宇付丽娜袁玉玉丛聪耿慧君赵红
Owner DALIAN UNIV OF TECH
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