High-throughput Screening Platform For Evaluating The In Vivo Antimicrobial Activity Of Antipyretic-antidotal Traditional Chinese Medicine

Objective:In recent years Acinetobacter baumannii has been prevalent worldwide and has become one of the most important nosocomial pathogens.Especially for Extensively drug resistant Acinetobacter baumannii（XDRAB）,the clinical treatments become very difficult because of the rapid dissemination of drug resistance and the multi-mechanisms of drug resistance.Therefore,it is quite urgent to develop novel antibiotics.Traditional Chinese Medicine（TCM）has been used in the treatment of infectious diseases in China for a long time and has accumulated rich experience,finding antibacterial compounds in antipyretic-antidotal TCM has been thought to be an effective way.Nematode infection model can reflect the drug activity in vivo,screen antibacterial compounds efficiently and many other advantages.It is an ideal experimental model for antimicrobial screening.We had established an infection model of XDRAB-C.elegans to screen antibiotics in the early preliminarily stage,which was used in the screening of Chinese traditional medicine with antimicrobial activity.However,the model was not perfect and the scope of screening TCM was not wide enough.Therefore,this study aims to improve the reliability and stability of nematode infection model by optimizing the conditions of the model.Based on this model,the antibacterial activity of many kinds of antipyretic-antidotal TCMs were evaluated to find the TCM with antibacterial activity in vivo.Then the antibacterial activity of the main components of active TCM in vivo and in vitro was further evaluated to provide scientific basis for the research and development of new and efficient antimicrobial agents.Methods:1.To establish the XDRAB-C.elegans infection model,glp-4（bn2ts）;sek-1（km4）Caenorhabditis elegans was chosen as the animal model.By observing the survival rate and survival state of nematodes,we optimized the medium composition,bacterial load,infection duration of XDRAB,and the intervention time of drugs to construct an in vivo antibacterial model.2.2.1 Medicinalmaterialsextraction:Wesort out73antipyretic-antidotal TCM from Chinese Pharmacopoeia 2010 edition,extractions will be conducted utilizing water,50%ethanol and 95%ethanol as solvents by refluxing extraction.liquor ratio is 1:10,extracted2 times,each time 1h,merged the extract liquid and then dried in water bath pot to get extract,then stored at-20℃.2.2 Antibacterial activity evaluation:The different extracts of traditional Chinese medicine were applied to the XDRAB-C.elegans infection model to screen the antibacterial activity in vivo,each extract was diluted to five concentrations（1000μg/mL,100μg/mL,10μg/mL,1μg/mL,0.1μg/mL）,the antimicrobial activity of nematodes was evaluated according to the survival rate of nematodes.3.The main components of extracts with good antibacterial activity were analyzed and compared in vivo and in vitro by nematode infection model and broth dilution method.Results:1.In the study,different concentrations of XDRAB and Fe³⁺led to different survival rate of the C.elegans,5×10⁶ CFU/mL of XDRAB with10μmol Fe³⁺were selected to infect C.elegans in 20%BHI.Different infection time and treatment time are the key factors for model construction,Log-rank test results showed that survival curves of C.elegans infected with XDRAB 3 hours and vehicle control were not significantly different（χ²=3.154,P>0.05）;survival curves of C.elegans infected with XDRAB 6 hours and vehicle or PB control were significantly different（both P<0.001）;survival curves of C.elegans infected with XDRAB 6 hours and 9 hours were not significantly different（χ²=0.669,P>0.05）;so 6 h was chosen as infection time and 36h as the appropriate time for antimicrobial therapy.2.XDRAB-C.elegans model was used to evaluate the antibacterial activity of the extracts from 73 antipyretic-antidotal TCM in vivo,the cortex moutan was screened with obvious antibacterial activity in vivo.3.The four main active components of cortex moutan,paeonol,paeoniflorin,betulinic acid and gallic acid were verified the antibacterial activity combined with XDRAB-C.elegans infection model and broth dilution method in vivo and in vitro.Paeonol,the main active component of cortex moutan,could improve the survival rate of XDRAB infected nematodes and had no toxicity to nematodes especially;in vitro antimicrobial test,the MIC of the paeonol was 1024μg/mL,the MIC of paeoniflorin was 2048μg/mL,gallic acid and betulinic acid have no effective antibacterial activity.Conclusion:In this study,XDRAB-C.elegans infection model was optimized,and extracts of the Chinese medicine cortex moutan with obvious antibacterial activity in vivo was screened from 73 antipyretic-antidotal TCM based on model,and it was verified that main active component paeonol of cortex moutan had obvious antibacterial activity in vivo and in vitro,which provide scientific basis for the research and development of new and efficient antimicrobial agents.