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Method for degrading streptococcus suis biofilm by applying phage lyase

A technology of phage lyase and biofilm, which is applied in the field of phage lyase to degrade Streptococcus suis biofilm, can solve the problems such as difficult to degrade Streptococcus suis, and achieve the effect of high-efficiency lysis

Active Publication Date: 2011-09-28
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] After searching the prior art, Daniel Grenier, Louis Grignon, and Marcelo Gottschalk published "Characterisation of biofilm formation by a Streptococcus suis meningitis isolate" on pages 292-295 of Volume 179 of "The Veterinary Journal" in 2009. And, the Streptococcus suis type II strain 95-8242 isolated from meningitis pigs can form a dense biofilm, and the resistance of Streptococcus suis 95-8242 in the biofilm to penicillin G and ampicillin is much stronger than that of Free bacteria, conventional drugs are difficult to degrade Streptococcus suis biofilm

Method used

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  • Method for degrading streptococcus suis biofilm by applying phage lyase
  • Method for degrading streptococcus suis biofilm by applying phage lyase
  • Method for degrading streptococcus suis biofilm by applying phage lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cleavage spectrum of biofilm by lyase:

[0033] Step 1, cultivation of biofilm

[0034] Inoculate 20 μL of Streptococcus suis overnight on a shaker at 37°C into each well containing 2 mL of THB medium (add 3 g of yeast extract, 20 g of tryptone, 5 g of beef extract, 2 g of glucose, 10 g of NaCl, and 2 CO 3 2.5 g, 1.0008 g of disodium hydrogen phosphate, 20 mL of calf serum, adjust the pH value to 7.5) in a 24-well cell culture plate (purchased from Cellstar Company), cover and culture in a 37°C incubator for 3 days. Wells containing only THB medium were used as control wells. After 3 days, the cell culture plate was taken out, the free medium was discarded with a pipette and washed gently twice with sterilized PBS buffer (pH7.2) to remove free bacteria, and a mature biofilm was obtained.

[0035] Step 2, expression and purification of phage lyase LySMP

[0036] The plasmid used for the prokaryotic expression is pET-28a(+), and the expressed cleavage enzyme molecule...

Embodiment 2

[0052] The dose-effect relationship of lyase cleavage biofilm:

[0053] Step 1, dilute the purified LySMP with elution buffer to 50IU / mL, 100IU / mL, 200IU / mL, 400IU / mL, 800IU / mL. Add 0.5 mL of LySMP at different concentrations to the biofilm, that is, add 25 IU, 50 IU, 100 IU, 200 IU, and 400 IU to each well.

[0054] Step 2: After using LySMP to act on the biofilm for 6 hours in a 37° C. incubator, use the crystal violet staining method (same as Example 1) to semi-quantitatively measure the biofilm residual amount.

[0055] Such as figure 2 As shown, 25IU lyase has almost no cracking effect on biofilm. When it is greater than 50IU, the cleavage effect of the lyase is stronger.

Embodiment 3

[0057] The number of residual viable bacteria after the lyase acts on the SS2-4 biofilm:

[0058] Step 1, make MTT colorimetric test standard curve according to MTT colorimetric test method

[0059] In step 2, the Streptococcus suis biofilm treated with LySMP for 1, 2, 4, 6, 8, 12, 18, and 24 hours was gently washed twice with sterile PBS buffer (pH 7.2) to remove free bacteria. Resuspend residual biofilm in 1 mL of sterile PBS buffer per well.

[0060] Step 3: Use a pipette to draw 100 μL of the bacterial resuspension, and add it to a 96-well microtiter plate, and make 3 replicate wells for each sample solution, and set a negative control with PBS buffer. Then add 20 μL of MTT application solution (100 mg MTT in 20 mL PBS buffer solution, MTT purchased from sigma company) to each well, put it in a constant temperature incubator at 37 °C for 2 hours, take it out, and add 100 μL of DMSO (purchased from sigma company), the OD value was measured at 570nm with an automatic micro...

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Abstract

The invention provides a method for degrading a streptococcus suis biofilm by applying phage lyase, belonging to the field of biotechnology. The method comprises the following steps of: expressing lyase LySMP of which the molecular weight is 55kDa by adopting expression bacteria BL21-lys, purifying with a Ni column to obtain lyase, and degrading the streptococcus suis biofilm obtained by culture on a cell culture plate by using the lyase in vitro. The method provided by the invention can sterilize streptococcus suis SS2-4 and SS2-H and can simultaneously destroy the structure of the biofilm to achieve the effect on thoroughly clearing the biofilm.

Description

technical field [0001] The invention relates to a method in the field of biotechnology, in particular to a method for degrading Streptococcus suis biofilm by using phage lyase. Background technique [0002] Streptococcus suis is a zoonotic infection that can cause meningitis, septicemia, arthritis, endocarditis, pneumonia in piglets and meningitis in humans. Streptococcus suis type 2 is the most prevalent and the most pathogenic to pigs, causing huge economic losses to the pig industry and posing a serious threat to the lives of relevant practitioners in terms of public health. What's more, when S. suis forms a biofilm, the biofilm provides the bacteria with a protective lifestyle that may greatly increase its pathogenicity. Bacterial biofilm refers to the structural community of bacterial cells wrapped by extracellular polysaccharide matrix, lipoprotein, fibrin, etc. produced by the bacteria themselves. The formation of bacterial biofilm enhances the resistance of bacteri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/51A61P31/04
Inventor 孙建和孟祥朋严亚贤陆承平张静
Owner SHANGHAI JIAO TONG UNIV
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