In Vitro Reversal Study On The Resistance Of Berberine To Clinical Isolates Of Carbapenem-producing Klebsiella Pneumoniae Isolates

PURPOSECarbapenem antibiotics are the last line of defense against bacterial infections.However,with the increase in clinical use,drug-resistant strains have emerged in recent years,and we found that our hospital is resistant to carbapenem-resistant Klebsiella pneumoniae(CRKP)within five years.The detection rate climbed from 0.46% to 5.27%,and the drug resistance situation is not optimistic,which should cause us to pay enough attention.This subject investigated the status and mechanism of CRKP resistance in our hospital,identified its drug resistance genotype,and analyzed ST type,and further selected the Chinese medicine monomer berberine to act on Kpneumoniae carbapenemase(KPC).The strains were observed to reverse the drug resistance,providing a laboratory basis for the prevention and treatment of drug-resistant bacteria.METHODWe intend to retrospectively analyze the bacterial counts of Enterobacteriaceae in the Second Affiliated Hospital of Henan University of Traditional Chinese Medicine from 2015 to 2017,calculate the detection rate of CRKP,remove the same patient repeat strain,resuscitate selected strains,and use VITEC2-Compact automatic microorganisms.The analyzer re-analyzed the strains and reviewed the drug susceptibility results,and analyzed the resistance to common antimicrobial drugs using WHONET 5.6 software.The metalloenzyme phenotype was initially screened by EDTA-imipenem double-paper synergy method.The drug resistance genotypes were detected by PCR and agarose electrophoresis.Multilocus sequence typing(MLST)was used to determine the strains.The kinship of existence.Then,Escherichia coli EC600(sensitive to meropenem,rifampicin resistant)was selected as the recipient bacteria,and the KPC-producing CRKP was identified as the donor bacteria.The junction transfer experiment was carried out to investigate whether the drug-resistant gene has the property of spreading.The preparation contains 2μg/ml MEM,100 μg/ml rifampicin Chinese blue screening plate was screened for zygote,the binding efficiency was counted,and the zygote was subcultured continuously.After 5 days,the resistance to MEM was reviewed by paper method to explore the junction.The stability of the plasmid.Finally,the traditional Chinese medicine monomer berberine was applied to CRKP,and the MIC of berberine to CRKP was determined by micro-broth dilution method.The sub-MIC concentration of berberine and CRKP were co-cultured with 1% SDS(dodecyl sulfate).Sodium was used as a positive control.After 24 h,48h and 72 h,the colonies were photocopied to Chinese blue screening plate and Columbia blood plate containing 2μg/ml MEM,and the elimination rate was determined to investigate the resistance of berberine to CRKP reversal.RESULTFrom January 2015 to August 2017,10222 bacterial infection samples were detected,including 1166 cases of Klebsiella pneumoniae,accounting for 11.4%,82 cases of CRKP by non-repetitive separation,and 82 strains of CRKP showed higher resistance rate.Resistance or mediation of most antibacterial drugs is only relatively sensitive to minocycline,tigecycline and fosfomycin,which are less clinically used.Among the 82 strains of CRKP,23 strains produced ESBLs,the ratio was 28.04%(23/82),and 20 strains of 82 strains of CRKP were detected by EDTA-imipenem double-paper synergistic method.The results of drug resistance gene test showed: There were 64 strains of KPC positive.The MLST results showed that the ST11 type was the most,with 21 strains.A total of 19 KPC-producing CRKP strains were selected for plasmid junction transfer assay.Six of them were successfully joined,accounting for 31.58%(6/19).They were still resistant to carbapenems after 5 days of continuous seeding.The R plasmid elimination rates of berberine against the test strains at 24 h,48h and 72 h were: 0,3.1% and 4.7%,respectively;sodium dodecyl sulfate(SDS)eliminated the R plasmid of the test strains at 24 h,48h and 72 h.The rates were 1.1%,4%,and 8.1%,respectively;the blank elimination control rate was zero for all time periods,thus eliminating the possibility of plasmid self-loss in replication.CONCLUSIONThe detection rate of CRKP is increasing year by year.It is a serious problem.To attract enough attention,the most important mechanism of CRKP isolation in our hospital is carbapenemase.High-level drug resistance can be combined with other ESBLs-producing mechanisms.The sequence of CRKP multi-site sequence showed mainly ST11 type,accounting for 63.6%,suggesting that the resistant bacteria have certain spread in the hospital.The main drug resistance genotype in our hospital is KPC.Some KPC can be transmitted through the plasmid.Although the probability of transmission is not high,it is worth noting that the drug-resistant plasmid has genetic stability.The hinese medicine berberine has low MIC for CRKP.The antibacterial effect,and the sub-MIC concentration of berberine has a certain reversal effect on the resistance of CRKP.Experimental studies have found that the reversal rate has no SDS effect,but SDS is a chemical reagent and cannot be used in clinical practice.