Method for screening drug which is resistant to pan-drug-resistant acinetobacter baumannii by using caenorhabditis elegans

A technology for Acinetobacter baumannii and Caenorhabditis elegans, which is applied in the biological field and achieves the effects of low preparation cost, simple operation and short cycle

Active Publication Date: 2018-03-06
GUANGZHOU GENERAL HOSPITAL OF GUANGZHOU MILITARY COMMAND
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  • Description
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  • Application Information

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Problems solved by technology

[0004] At present, the research on EPIs is mostly limited to in vitro experiments. Whether different EPIs can reverse the drug resistance of Acinetobacter baumannii in vivo to achieve a better antibacterial effect, and whether they will have a greater toxic effect is the current application of EPIs. Major issues that need to be resolved through in vivo experimental studies

Method used

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  • Method for screening drug which is resistant to pan-drug-resistant acinetobacter baumannii by using caenorhabditis elegans
  • Method for screening drug which is resistant to pan-drug-resistant acinetobacter baumannii by using caenorhabditis elegans
  • Method for screening drug which is resistant to pan-drug-resistant acinetobacter baumannii by using caenorhabditis elegans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Establishment of Caenorhabditis elegans-pan-drug-resistant Acinetobacter baumannii infection model

[0042] 1. Test material

[0043] (1) Caenorhabditis elegans used in this example ( glp-4;sek-1 ), Escherichia coli ( E. coli ) OP50 was donated by Professor Chen Yanguang, School of Pharmacy, Sun Yat-sen University. The quality control strain Escherichia coli ATCC25922, and the clinically isolated pan-drug-resistant Acinetobacter baumannii (XDR-AB) were collected from ICU patients by the Laboratory Department of Guangzhou General Hospital of Guangzhou Military Command. Mérieux's VITEK-2 microbial automatic identification drug susceptibility instrument identification.

[0044] (2) CCCP (Carbonyl cyanide 3-chlorophenyl hydrazone), PAβN (phenyl-arginine-β-naphthylamide), polymyxin B (Polymyxin B sulfate salt), nalidixic acid, verapamil hydrochloride), reserpine (Reserpine), and omeprazole (Omeprazole) were purchased from Sigma Chemical Reagent Company of the ...

Embodiment 2

[0066] Example 2 Toxicity test of efflux pump inhibitors

[0067] 1. Wash the synchronously cultured nematodes to L4 stage with 20% BHI liquid medium into a 15mL centrifuge tube, wash three times at 500 r / min, 1min, and distribute 15-20 strips / well into a 96-well plate to 180μL, Add serial concentrations of efflux pump inhibitor CCCP, PAβN (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL), NMP, Austrian Meprazole, verapamil, reserpine (10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL) 20 μL, at 25°C 1. Under the condition of 85% humidity for 30 hours without co-cultivation, the survival rate of nematodes was observed under a microscope, and the toxic effect of each efflux pump inhibitor on normal nematodes was judged.

[0068] 2. Results

[0069] The six efflux pump inhibitors CCCP, NMP, PAβN, omeprazole, verapamil and reserpine have different toxic effects on nematodes. Among them, when the concentration of CCCP is...

Embodiment 3

[0070] Example 3 In vitro drug susceptibility test of ciprofloxacin and various efflux pump inhibitors

[0071] 1. Microbroth dilution method to determine the minimum inhibitory concentration (MIC) of antibacterial drug ciprofloxacin

[0072] Escherichia coli ATCC25922 and Acinetobacter baumannii ATCC19606 were used as quality control strains for testing in the same batch. Prepare 5120 μg / mL antibacterial drug stock solution, take 13 sterile test tubes, and use CAMHB medium to prepare series concentrations of ciprofloxacin hydrochloride 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL , 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, take 100 μL each In a 96-well plate; use a photometric turbidimeter to take freshly cultured colonies and adjust the concentration of the bacterial suspension to 0.5 McFarland units (about 1.5×10 8 CFU / mL), 100 μL was added to each well to make the final concentration of bacteria 5×10 6 CFU / mL, the concent...

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Abstract

The invention discloses a method for screening a drug which is resistant to pan-drug-resistant acinetobacter baumannii by using caenorhabditis elegans. First, a caenorhabditis elegans-pan-drug-resistant acinetobacter baumannii infection model used for screening the efficacy of a composition is constructed, wherein caenorhabditis elegans has double gene mutation of glp-4 and sek-1, the culture medium of the caenorhabditis elegans-pan-drug-resistant acinetobacter baumannii co-culture consists of 20% of BHI, 5-20[mu]m of nalidixic acid and 5-20[mu]g / ml of FeCl3; the concentration of the pan-drug-resistant acinetobacter baumannii is 1*10<6>-1*10<9>CFU / mL; the duration time of the bacterial infection on the caenorhabditis elegans is 6-12 hours; and the time of the treatment on an infected modelby a drug is 24-48 hours. The model can be used for fast and high-throughput screening of in-vivo antibacterial activity of various compounds or drugs or compositions, and compared with an in-vivo animal infection model, the model has the huge advantages of low preparation cost, short period and easy operation. Compared with an in vitro model, the model can be used to screen out compounds which have large in-vivo toxicity, poor metabolism and low in vitro-in vivo correlation.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a method for screening drugs against pan-drug-resistant Acinetobacter baumannii by using Caenorhabditis elegans. Background technique [0002] Acinetobacter baumannii ( Acinetobacter baumannii ) is a non-fermenting Gram-negative bacillus that widely exists in nature and hospital environments, and is one of the most common opportunistic pathogens causing nosocomial infections. Acinetobacter baumannii can cause a variety of infectious diseases including respiratory tract infection, urinary system infection, respiratory tract infection, bacteremia, wound infection, meningitis and ventilator-associated pneumonia, etc. The widespread use of the bacteria has produced great resistance to commonly used antibiotics, and even multi-drug resistance (resistance to 3 or more types of commonly used antibacterial drugs) Acinetobacter baumannii (MDR-AB) and extensively drug-resist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/02A61K49/00
CPCA01K67/02A61K49/0008
Inventor 姜志辉李健段欣冉何羡霞
Owner GUANGZHOU GENERAL HOSPITAL OF GUANGZHOU MILITARY COMMAND
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