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Method for rapidly determining antibiotic resistance of Escherichia coli in biofilm

A technology of antibiotic resistance and biofilm, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., to achieve the effect of easy repeatability, less supporting equipment, and simple operation

Inactive Publication Date: 2018-12-14
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there has been no research on the resistance of E. coli to colistin in biofilms.

Method used

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  • Method for rapidly determining antibiotic resistance of Escherichia coli in biofilm
  • Method for rapidly determining antibiotic resistance of Escherichia coli in biofilm
  • Method for rapidly determining antibiotic resistance of Escherichia coli in biofilm

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A method for constructing Escherichia coli in a biofilm resistant to polymyxins, the experimental flow chart is as follows figure 1 As shown, the order is: the activation of the strain; the biofilm growth stage; the antibiotic stress stage; the biofilm recovery stage. The steps of the method include:

[0033] (1) Biofilm growth stage

[0034] Take out the Escherichia coli stored in the glycerol tube from the -80°C refrigerator, place it on the ice box, and thaw it for about 5 minutes. In the aseptic operating table, use a sterile inoculation loop to draw a line on the LB agar plate, place it in a constant temperature incubator, and incubate at 37°C for 16-18h. Pick a single colony and put it in 9mL LB broth, place it in a shaker at 37°C and 200rpm and culture it for 16-18h.

[0035] Centrifuge the cultured thalline at 3000rpm, 10min, and 25°C, remove the supernatant (the supernatant is the centrifuged medium), and adjust the concentration of the bacterial solution wi...

Embodiment 2

[0056] Embodiment 2 plate coating method determines Escherichia coli optimal culture temperature

[0057] According to the above-mentioned method of activating and diluting, the OD value of the thalline was adjusted to the bacterium solution of OD600 value=0.5 McFarland, (the bacterial concentration was about 1×10 9 cfu / mL) for the cultivation of biofilms. Take 10 μL of the above-mentioned E. coli culture solution and add it to a 24-well plate containing 990 μL fresh LB (3% NaCL) medium, and use the fresh LB well plate not inoculated with the bacteria solution as a blank control, and make 3 parallels for each strain. Select four different temperatures (15°C, 25°C, 37°C and 45°C) and culture them statically for 24 hours to measure the amount of biofilm formation. The 24-well plate was sealed with plastic wrap to prevent moisture from evaporating.

[0058] After the cultivation, the bacterial solution in the well plate was first discarded, and the non-adhered bacteria were was...

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Abstract

The invention belongs to the technical field of biology and discloses a method for rapidly determining antibiotic resistance of Escherichia coli in a biofilm. The method comprises steps as follows: abiofilm growing stage; an antibiotic pressure stage; a biofilm recovery stage. According to the method, an in-vitro model of Gram negative bacteria such as Escherichia coli biofilm is established by adopting a micro-inoculation needle as a carrier, and the method is used for measuring BIC (biofilm inhibitory concentration) and MBEC (minimal biofilm eradication concentration) of antibiotics such aspolymyxin for the Escherichia coli biofilm. After the biofilm is formed for microorganisms, the degree of resistance to colistin is increased, BIC and MBEC values are both increased, and the MBEC value is larger than the BIC value. Compared with other methods, the in-vitro forming experiment of the Escherichia coli biofilm in the research has the characteristics of being simple to operate, easy to repeat and the like; meanwhile, few support devices are required, technical requirement is low, and errors caused by human factors and the like can be reduced; the method is applicable to larger samples; theoretical basis is provided for clinical rational drug use.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for quickly detecting the antibiotic resistance of Escherichia coli in a biofilm. Background technique [0002] Antibiotic resistance is a huge challenge in global public health. Globally, there are 700,000 deaths caused by multidrug-resistant pathogens each year, and it is estimated that the number may reach 1 million in 2050. The serious problem of drug resistance makes human beings face serious challenges in the fields of medical clinic, veterinary clinic and food safety. At the same time, it is reported that about one million people around the world suffer from illness or death due to bacterial biofilm infection every year. After the bacteria form a biofilm, they have strong drug resistance and can evade the immune function of the host. It is difficult to completely remove the infection site, which is a difficult clinical One of the important causes of sexually transmitt...

Claims

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Application Information

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IPC IPC(8): C12Q1/18
CPCC12Q1/18
Inventor 赵勇白凤佳张昭寰牛丽刘海泉
Owner SHANGHAI OCEAN UNIV
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