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A method for artificially obtaining bacterial drug-resistant strains in vitro

A technology for drug-resistant strains and bacteria, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. Good for quick effect

Inactive Publication Date: 2020-03-24
BEIJING UNIV OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has certain limitations, which are mainly manifested in: ①The subinhibitory concentration of the stimulating bacteria is artificially set, different concentrations bring different experimental results, and the experimental conditions are not easy to control; Errors are prone to occur in the process, which has a great impact on the experimental results; ③A certain concentration of bacterial liquid must be prepared for each passaging, which is cumbersome and prone to errors; ④It is necessary to transfer the bacteria to Seed on a solid plate to observe the inhibition zone or measure the MIC, there are many experimental links; ⑤The induction period is long

Method used

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  • A method for artificially obtaining bacterial drug-resistant strains in vitro
  • A method for artificially obtaining bacterial drug-resistant strains in vitro

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1 material

[0026] Strains: Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC25922), passed to the second generation after resuscitation, and preserved at 4°C for future use.

[0027] Medium: nutrient agar, used for culturing and preserving bacteria; MHA (Mueller-Hinton Agar), used for detecting bacterial MIC; MHB (Mueller-Hinton Broth), used for induction of bacterial passage. The diameter of the plate is 9 cm, and the thickness of the medium is about 4 to 5 mm.

[0028] Antibiotic standard: detection of bacterial MIC. Dilute to 1 mg / ml with sterile PBS, and store at -20°C after aliquoting.

[0029] Drug-containing discs: commercially available imipenem-containing discs and ceftazidime-containing discs. It is the same species as the antibiotic standard, and it is stored at -20°C for future use.

[0030] 2 Experimental methods

[0031] Before the drug-containing disc induces a certain bacterium, the MIC of the bacterium ...

Embodiment 2

[0049] Embodiment 2 The comparison of the inventive method and broth subculture induction method

[0050] Broth passage induction method:

[0051] Experimental method: Inoculate 10 μl of the 0.5 McFarland concentration test strain bacterial liquid in 1 / 4 MIC antibiotic concentration nutrient broth, culture in a 35°C incubator for 24 hours as one generation, and then take the first generation bacterial liquid from the medium and dilute it as After the above concentration, continue to pass to the next same culture broth... Repeat the passaging in this way, and measure and record the diameter of the inhibition zone of each generation of transformed strains with the disk method at the same time, according to the Clinical Laboratory Standardization Institute (CLSI) Standards for judging the emergence of drug resistance. When drug resistance occurs, accurately measure the MIC value of this generation of bacteria. If the MIC value after induction is >4 times the MIC before induction...

Embodiment 3

[0064] Embodiment 3 preparation method of traditional Chinese medicine medicine-containing paper sheet

[0065] The method of the present invention selects commercially available standard drug-sensitive paper sheets for induction, and self-made drug-sensitive paper sheets can also be used for induction. The method of self-made drug-sensitive paper sheets is as follows:

[0066] Use a hole puncher to make Xinhua No. 1 qualitative filter paper into 6mm circular filter paper sheets, pack 50 pieces into a penicillin vial, seal with a single layer of kraft paper and sterilize under high pressure (121°C, 15-20min), and dry for later use .

[0067] Prepare traditional Chinese medicine (water decoction, extract or monomer) into a certain concentration of liquid medicine, put a certain amount of liquid medicine into a small bottle with 50 pieces of filter paper, so that the paper pieces are fully soaked in the liquid medicine, 37 ° C, 24 ~ 36h, dry and seal for later use. Keep dry or...

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Abstract

The invention provides a method for artificially obtaining a bacterial drug-resistant strain in vitro. The method comprises the following steps: a, preparing a carrier loaded with a to-be-tested drug, wherein the carrier is capable of absorbing moisture; b, detecting the minimum inhibitory concentration of the screened bacteria before culture; c, coating the screened bacteria on a plate medium, placing the carrier loaded with the to-be-tested drug on the surface of the culture medium, culturing the bacteria, and forming an inhibition zone on the carrier loaded with the to-be-tested drug; d, taking the bacteria in a septic area which is 0.5-4mm away from the edge of the inhibition zone obtained in the step c, and repeating the step c; and e, detecting the minimum inhibitory concentration of the bacteria cultured each time, and obtaining a drug-resistant bacterial model when the minimum inhibitory concentration of the cultured bacteria is detected to reach the minimum inhibitory concentration before culture before 4-times induction.

Description

technical field [0001] The invention relates to a method for artificially obtaining bacterial drug-resistant strains in vitro, belonging to the field of biology. Background technique [0002] With the widespread use of antibacterial drugs, drug-resistant bacteria continue to emerge, and bacterial drug resistance is also increasing. The continuous increase of drug-resistant bacteria has brought great difficulties to the treatment of human and animal diseases. The emergence and spread of bacterial drug resistance has attracted worldwide attention and is considered by the World Health Organization to be one of the biggest public health security problems in the 21st century. Therefore, the detection, monitoring and evaluation of bacterial resistance is very necessary and urgent. [0003] Bacterial drug resistance research methods include drug susceptibility experiments, plasmid elimination experiments and plasmid fingerprinting technology, gene probe technology, PCR technology...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04
CPCY02A50/30
Inventor 邱泽计吴珺高洁郝钰孔令博王旭丹
Owner BEIJING UNIV OF CHINESE MEDICINE