Virulence Characteristics And Transcriptomic Analysis Of Imipenem-induced Klebsiella Pneumoniae Resistance In Vitro

Objective:To induce Hypervirulence Klebsiella pneumonia strain resisitant to imipenem in vitro.The physiological changes of the induced strains ? the drug resistance and virulence characteristics and the ability of biofilm formation were analyzed.The causes of those changes were analyzed by RNA-sequencing.Methods:1.The highly virulent Klebsiella pneumoniae strains that have been identified have been induced using a low-concentration broth induction method.After induction,the MIC of drug-resistant strains were screened by E-test strips.16 S RNAseque-ncing and PFGE homology analysis were performed to identify whether the strains which induced by imipenem before and aftre were consistent.2.Observe the physiological changes of induced drug-resistant strains: hyperm-ucoviscosity phenotype detection,PCR detection of drug resistance genes and virulence genes,CPS quantification.3.The changes of virulence characteristics of the induced strains were detected by serum resistance,anti-neutrophil phagocytosis experiments,evaluate the ability of induced strains biofilm formation and mouse virulence experiments,and the expression of related virulence genes was analyzed by transcrip-tomics.Results:1.Hypervirulence Klebsiella pneumoniae were induced by imipenem in vitro to produce drug-resistant strains with MIC values of 2?4 respectively.After the analysis of 16 S RNA sequencing and PFGE homology,the three strains before and after induction were homologous to Klebsiella pneumoniae.2.Compared with the hypervirulent Klebsiella pneumoniae,a series of microbiological changes occurred in the resistant strains with MIC values of 2,4 after induction: the disappearance of wire drawing and PCR detection of virulence genes such as rmpA,terW,silS was negative and the biosynthesis of CPS was reduced.The serum resistance and phagocytic capacity of anti-neutrophil were decreased in different degrees.The virulence results in mice showed that the lethal rate of drug-resistant strains after induction decreased in mice.The biofilm formation capacity of the induced strains was increased.In transcriptomic analysis,of the 243 virulence-related genes were changed,102 genes were down-regulated and 141 genes were up-regulated.Of the 65 drug resistance-related genes,26 genes were down-regulated and 39 genes were up-regulated.Compared with the pre-induction hypervirulent Klebsiella pneumoniae,there were 10 genes with an MIC value of 2 Down-regulated expression,up-regulated expression of 3 genes among the 24 genes involved in CPS synthesis,;15 genes with down-regulated expression of MIC and 4 genes,and up-regulated expression of 3 genes among the 24 genes involved in CPS synthesis,.The expression levels of the gene wcaG,galF,gmd,and wzy were significantly decreased in the strains with MIC values of 2 and 4 after induction.Conclusion:In view of this,we have discovered that the virulence of K.pneumoniae has undergone major changes in its resistance.Based on the above results,we speculate that the loss of part of the virulence genes and the down-regulation of the expression of CPS synthesis genes in the course of drug resistance in Klebsiella pneumoniae with high virulent strains which may change the virulence of the bacteria(such as serum Resistance,neutrophil phagocytosis,and lethality in mice)play an important role.