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
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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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