Study On The Difference Of Drug Resistance Caused By Different KPC Subtypes And Its Molecular Mechanism

Purpose:Klebsiella pneumoniae Carbapenemase（KPC,Klebsiella pneumoniae Carbapenemase）,it is one of the most importantβ-lactamases,now it has appeared many subtypes due to mutations,and gradually developed into a big family,the result of these subtypes is drug resistance.In this study,we mainly construct KPC2-36 clones with the same genetic background and conduct drug susceptibility experiments to explore their evolutionary trends and differences in drug resistance caused by different subtypes.Finally,the main mechanism of drug resistance caused by its subtypes was verified by enzyme activity experiments and molecular structure.Method:In order to study the evolution trend and drug resistance differences of the subtypes of Klebsiella pneumoniae carbapenemase,we used MAGE software to count the different subtypes,and constructed the KPC phylogenetic tree using the maximum likelihood method.At the same time,we constructed a total of 44 recombinant plasmids of p ET28a-Tn4401-KPC2-36,including 8 unnatural mutants,and transformed the resulting recombinant plasmids into Escherichia coli to have the same genetic background.In the sensitivity test,we tested the minimum inhibitory concentration values of all KPC2-36 strains.In order to further verify the ability of these strains to hydrolyze drugs,we constructed the expression vector plasmid p ET28a-KPC-X to expressing and purifying the KPC recombinant protein.Enzyme activity experiment finally obtained steady-state kinetic parameters of different KPC recombinant proteins.In order to study whether avibactam inhibits KPC enzyme,we also used ITC experiments to verify the molecular mechanism.Results:We performed a systematic reconstruction of KPC subtypes,and divided all subtypes into KPC-2 system group and KPC-3 system group according to the H274Y mutation site,except for bla KPC-2 and bla KPC-3,which were generated in 40 total changes There were 25single mutations,8 double mutations and 7 multi-site mutations,and the region with higher mutation frequency was between 200-250 amino acids,among which P103,F206,V239,and W104 were high-frequency mutation sites.The drug susceptibility test found that almost all（20/22）KPC-3 variants are not sensitive to ceftazidime（CAZ）,and mutations at the same site will have different drug resistance.In addition,there are 4wild-type KPC-3 variants.Type KPC（KPC-14,-15,-25 and KPC-31）and 3 artificial KPCs（KPC-3like2,KPC-8like1 and KPC-15like2）,The minimum inhibitory concentration（MIC）of ceftazidime/avibactam（CAZ/AVI）has reached the effective concentration MIC90 value proven in the literature,where CAZ is 2 mg/L and avibactam（AVI）is 4 mg/L.Although KPC can hydrolyze aztreonam（ATM）,aztreonam/avibactam（ATM/AVI）can effectively inhibit more than 90%of large intestine angstroms that carry KPC（≤0.5 mg/L）.The growth of Greek bacteria.Enzyme activity experiments showed the hydrolysis ability of each subtype to various drugs,and it was found that compared with KPC-2,the hydrolysis of KPC-4,-14,-28,-31,etc.was enhanced.The ITC experiment found that KPC-2 and KPC-3 have higher affinity for AVI,while in comparison,KPC-14,-28,-31 have lower affinity for AVI.Conclusion:The diversified development of bla_KPC-2 and bla_KPC-3nodes is mainly the result of a single mutation.For bla_KPC mutations with a single non-synonymous mutation,there is only one evolutionary situation,and it is indisputable.Secondly,changes in amino acids may change the resistance of strains to drugs.Even if the mutation is at the same site,the different amino acids that are mutated will make the strains have different resistance changes.In the evolution of KPC,CAZ may be the main one.we verified the ability of each subtype to hydrolyze drugs in vitro and also proved that CAZ is the driving factor for the evolution of KPC tested by enzyme activity experiments,.At the same time,among those strains resistant to CAZ,we found that some strains can resist the treatment of CAZ/AVI.ITC experiments revealed that the inhibition of KPC enzyme by AVI can restore the antibacterial activity of ceftazidime,and it also confirmed that some strains have CAZ.The reason for the increase of CAZ/AVI resistance is mainly due to the weakening of AVI inhibition of KPC,and the increase of CAZ hydrolysis of KPC.Later,in the determination of the strain-sensitive and drug-resistant mediators,we found that ATM/AVI is a viable treatment method for inhibiting KPC subtype strains.We provide a detailed research report on the resistance ofβ-lactam antibiotics to KPC2-36,and expand the activity spectrum of some special subtypes on the substrate.For the first time,the ITC experiment was used to verify and explain the changes in the resistance of CAZ/AVI.Conducive to the study of resistance mechanisms.