Preliminary Study On Antibacterial Sensitization Effect Of Artesunate Against Carbapenem-resistant Klebsiella Pneumoniae And Its Mechanism

Objective: Clinically,the isolation rate of Klebsiella pneumoniae ranks second,after Escherichia coli.In recent years,the resistance rate of Klebsiella pneumoniae to carbapenem antibiotics has been increasing year by year,which presents new challenges to the treatment of clinical infectious diseases.Therefore,it is urgent to find effective antibacterial drugs to solve clinical problems is the core content of this study.As an antimalarial drug,artesunate also has a certain pharmacological effect in other infectious diseases.This study aims to investigate the antibacterial sensitization effect of artesunate or its combined antibacterial agents on carbapenem-resistant Klebsiella pneumoniae and its possible mechanism in order to evaluate the potential clinical application value of artesunate and provide experimental evidence for the treatment of carbapenemresistant Klebsiella pneumoniae by artemisinin drugs.Methods:1.Collect the clinical data of Klebsiella pneumoniae isolates from Changsha Central Hospital affiliated to University of south China in 2018,and use WHONET software to statistically analyze its source,distribution and drug resistance.Thirty-two carbapenem-resistant Klebsiella pneumoniae strains were collected and stored from them;2.The MTT colorimetric method was used to detect the activity of different concentrations of drugs on carbapenem-resistant Klebsiella pneumoniae;3.The bacterial dynamic growth curve method was used to observe the effect of artesunate combine cephalosporin and other drugs on the growth of carbapenem-resistant Klebsiella pneumoniae;4.Using micro broth dilution method to detect the antibacterial effect of artesunate alone or in combination with cephalosporin and other drugs on carbapenem-resistant Klebsiella pneumoniae;5.Observe the antibacterial effect of artesunate combined with cephalosporin and other drugs on carbapenem-resistant Klebsiella pneumoniae by bacterial colony counting method;6.qRT-PCR was used to detect the expression level of efflux pump gene AcrB mRNA and the expression levels of its regulating genes MarA and AcrR mRNA;7.Use Excel 2016 and SPSS 25.0 software for data collation and statistical analysis.P < 0.05 was considered as a difference and had statistical significance.Results:1.The clinical isolates of Klebsiella pneumoniae mainly come from the pulmonary/respiratory ward,accounting for 39.22%,mainly from respiratory specimens,accounting for 75.35%.The resistance rate of Klebsiella pneumoniae strains to mezlocillin was 66.8%,and the resistance rate to tigecycline was 0.8%.Carbapenem-resistant Klebsiella pneumoniae accounted for 13.8% of clinical isolates of Klebsiella pneumoniae;2.The dynamic growth curve of bacteria shows that when 2048 μg/mL artesunate used alone,there is no obvious bacteriostatic effect on the growth of carbapenem-resistant Klebsiella pneumoniae,and the curve shows a clear upward trend.When artesunate is used in combination with ceftriaxone sodium,ceftazidime,ciprofloxacin,and amikacin,it can obviously inhibit the growth of carbapenem-resistant Klebsiella pneumoniae,and the curve is gentle.When artesunate was combined with meropenem,imipenem,and levofloxacin,there was no obvious bacteriostatic effect compared with the antimicrobial groups alone,and the curve showed an upward trend;3.The results of artesunate combined with antibacterial drugs showed that 2048 μg/mL artesunate can make ceftriaxone sodium,ceftazidime,ciprofloxacin,amikacin the MIC value to carbapenem-resistant Klebsiella pneumoniae was significantly reduced,and the combined effect evaluation was mainly based on the additive effect.When artesunate was used in combination with meropenem,imipenem and levofloxacin,there was no significant difference in the MIC value of carbapenem-resistant Klebsiella pneumoniae compared with the drug alone groups;4.The qRT-PCR results showed that among the 12 strains of carbapenem-resistant Klebsiella pneumoniae,4 strains had significant differences in the expression of AcrB gene between groups,2048 μg/mL artesunate combined with 256 μg/mL ceftriaxone sodium,2048 μg/mL artesunate combined with 1024 μg/mL ceftazidime significantly inhibited the expression of AcrB gene and the positive regulator MarA,but had no significant effect on the negative regulator AcrR expression;8 strains had no significant difference in AcrB expression between the groups.Conclusions:1.Klebsiella pneumoniae strains have the highest resistance to mezlocillin and the lowest resistance to tigecycline;2.Artesunate has no significant antibacterial effect on carbapenemresistant Klebsiella pneumoniae when it acts alone;3.Artesunate can significantly increase the sensitivity of carbapenemresistant Klebsiella pneumoniae to ceftriaxone sodium,ceftazidime,ciprofloxacin,and amikacin.Artesunate combined with meropenem,imipenem and levofloxacin have no obvious combined bacteriostatic effect;4.The combined antibacterial effect of artesunate combined with ceftriaxone sodium,and ceftazidime may be related to the inhibition of the expression of the efflux pump gene AcrB,and there may presence other mechanisms.