The Establishment Of Caenorhabditis Elegans Model Infection With Extensively Drug Resistant Acinetobacter Baumannii And Its Application In The Screening Of Antibiotics

Objective: Acinetobacter baumannii appear popular trendency all over the world and has been one of the most common pathogenic bacteria in china,due to its strong vitality and severe antibiotic resistance.However,research and development of new drugs can not keep up the pace with formation of drug-resistant strains,there will be a no drugs available situation.According to this case,many countries formulate preferential policies to encourage the development of antibiotics,hope that through this measure accelerate the speed of antibiotics development.Therefore,the development of antibiotics is not only significant,but also in line with international drug research and development trends.This paper intends to establish a Caenorhabditis elegans infection model of extensively drug-resistant Acinetobacter baumannii,and this model was used to screen heat-clearing and detoxifying herbs extract,in order to find anti-infective chinese herbs extracts.Methods: 1.To establish a Caenorhabditis elegans infection model of extensively drug-resistant Acinetobacter baumannii,extensively drug-resistant Acinetobacter baumannii clinical isolate GZLC-1 was choosen as the pathogen,glp-4(bn2ts);sek-1(km4)Caenorhabditis elegans was choosen as the animal model.The survival time of nematodes in Several liquid culture media(BHI broth,20% BHI–M9 buffer,10% BHI–M9 buffer,M9 buffer)with Acinetobacter baumannii clinical isolate GZLC-1 were compared to choose the suitable medium.In order to enhance the pathogenicity of Acinetobacter baumannii,we optimized the addition of iron ions in liquid medium.Finally,the composition of the liquid culture medium was determined.2.The optimal time of infection was determined by comparing the survival time of the infected worms.In addition,infection concentration of Acinetobacter baumannii and treatment time were optimized under optimal infection time.3.28 kinds of heat-clearing and detoxifying herbs was extacted by water,50% ethanol,95% ethanol,liquor ratio is 1:10,extracted 2 times,each time 1h,merged the extract liquid,drying into powder by water bath pot,the powder is kept in the 4℃ refrigerator.4.The Chinese herbal extracts were screened by Caenorhabditis elegans infection model of extensively drug-resistant Acinetobacter baumannii,each extract was diluted to five concentration(1000 μg/ml,100 μg/ml,10 μg/ml,1 μg/ml,0.1 μg/ml)for screening test,and at the same time established negative control group(1%DMSO)and positive control group(2μg/ml polymyxin B dissolved in 1%DMSO).5.The extracts with good antibacterial effect in the model were used for in vitro drug sensitivity test,and the antibacterial effects of extracts in vivo and in vitro were compared.Results: 1.The composition of the culture medium of the infection model was 10%BHI-M9,5μg/ml nalidixic acid,10μM Fe Cl3,1%DMSO.2.The infection time of the infection model is 6h,infection concentration of Acinetobacter baumannii is 5×105CFU/ml and duration of treatment is 24 h.3.Six water extracts(Radix Scutellariae,radix sophorae tonkinensis,Folium Isatidis,Cortex Moutan,Jigucao,Polygonum multiflorum)showed good antibacterial effect in vivo.4.The minimum inhibitory concentration(MIC)of six extracts(Radix Scutellariae,radix sophorae tonkinensis,Folium Isatidis,Cortex Moutan,Jigucao,Polygonum multiflorum)are larger than 2048μg/ml.Conclusion: 1.To establish a Caenorhabditis elegans infection model of extensively drug-resistant Acinetobacter baumannii,extensively drug-resistant Acinetobacter baumannii clinical isolate GZLC-1 was choosen as the pathogen,and we confirmed that the Caenorhabditis elegans were killed by Acinetobacter baumannii.2.The six kinds of Chinese herbal water extracts(Radix Scutellariae,radix sophorae tonkinensis,Folium Isatidis,Cortex Moutan,Jigucao,Polygonum multiflorum)have good therapeutic effect on the Caenorhabditis elegans which are infected by extensively drug-resistant Acinetobacter baumannii.3.The six kinds of Chinese herbal water extracts showed poor antibacterial effects in vitro,but good antibacterial effects in vivo,which tells us that these extracts are not direct sterilization,but to improve the immune system and other ways to improve the effectiveness of antibiotics.