Whole Genome Sequencing And Analyzing Of Extensively Drug-resistant Hypervirulent Klebsiella Pneumoniae XJ-K1 And XJ-K2 & The Preliminary Study Of Antibacterial Activity Of Five TCM Monomers Against Strains XJ-K1 And XJ-K2

Objective:The resistance characteristics,virulence characteristics,whole genome sequence and clonal type were analyzed on the clinical isolates of Klebsiella pneumoniae XJ-K1 and XJ-K2.Comparing the in vitro antibacterial activity of five traditional Chinese medicine monomers against strains XJ-K1 and XJ-K2 and analyzing the effect of combination of Chinese medicine monomers with better antibacterial activity and antibiotics.To provide scientific basis for cinical further understand the characteristics of Klebsiella pneumoniae,correct use of antibiotics and infection control of hospital,and also provide ideas for finding new treatment.Methods:1.Klebsiella pneumoniae strains XJ-K1 and XJ-K2 were isolated from the Northern Division of Huashan Hospital Affiliated to Hudan University.The minimum inhibitory concentrations of twenty-six antimicrobial agents against for Klebsiella pneumoniae XJ-K1 and XJ-K2were determined by broth microdilution method.2.The string test was used to determine the hypermucoviscous phenotype of strains XJ-K1 and XJ-K2.3.Resistance genes and virulence genes were amplified by PCR.4.Analysis of ST type of strains XJ-K1 and XJ-K2using MLST.5.The whole genome sequencing of strains XJ-K1 and XJ-K2 were performed by Illumina HiSeq 4000 and PacBio RSII.Genome assembly and gene prediction,and analysis of genome information by multiple bioinformatics analysis methods.6.The minimum inhibitory concentrations of five traditional Chinese medicine monomers（taxifolin,matrine,chlorogenic acid,luteolin and esculetin）against for Klebsiella pneumoniae XJ-K1 and XJ-K2 were determined by broth microdilution method.7.The drug combination between esculetin and six antimicrobial agents were performed by checkerboard dilution method,and fractional inhibitory concentration index（FICI）value was subsequently obtained by calculation.Results:1.Klebsiella pneumoniae XJ-K1 and XJ-K2 were resisted to all the tested antimicrobial agents except ploymycin B.2.The string test showed that strains XJ-K1 and XJ-K2 were hypermucoviscous.3.Resistance genes bla_TEM-1,bla_KPC,bla_SHV and rmtB were detected in strains XJ-K1 and XJ-K2;qnrS and tet（A）were detected only in strain XJ-K2.The known point mutations associated with quinolone resistance were found in gyrA and parC of strains XJ-K1 and XJ-K2.4.Strains XJ-K1 and XJ-K2 carried hypervirulent genes（iucABCD、iutA、rmpA and rmpA2）.5.The MLST showed that strains XJ-K1 and XJ-K2 belonged to ST11.6.（1）The Klebsiella pneumoniae XJ-K1 genome consists of a chromosome and three plasmids.The chromosome is 5,454,121bp in length and the three plasmids were 207,409bp（pXJ-K1-p1）,130,628bp（pXJ-K1-p2）and 99,048bp（pXJ-K1-KPC-2）in length.The strain XJ-K1 genome encodes thirteen resistance genes,and the chromosome of strain XJ-K1encodes three resistance genes（fosA,aadA2 and sul1）.The pXJ-K1-p2 encodes four resistance genes（dfrA12,mph（A）,aadA2 and sul1）,and the pXJ-K1-KPC-2 encodes six resistance genes(bla_TEM-1,bla_CTX-M-65,bla_SHV-12,bla_KPC-2,rmtB and fosA3).（2）The Klebsiella pneumoniae XJ-K2 genome also consists of a chromosome and three plasmids.The chromosome is 5,503,278bp in length and the three plasmids were 219,775bp（pXJ-K2-p1）,96030bp（pXJ-K2-KPC-2）and 84855bp（pXJ-K2-p3）in length.The strain XJ-K2genome encodes thirteen resistance genes,and the chromosome of strain XJ-K2 encodes three resistance genes(fosA,mdf（A）and bla_SHV-182).The pXJ-K2-KPC-2 encodes five resistance genes(bla_TEM-1,bla_CTX-M-65,bla_KPC-2,rmtB and fosA3),and the pXJ-K2-p3encodes five resistance genes(bla_LAP-2,dfrA14,qnrS1,sul2 and tet（A）-variant).（3）The chromosome and pLVPK-like virulence plasmid pXJ-K1-p1 of strain XJ-K1 and the chromosome and pLVPK-like virulence plasmid pXJ-K2-p1 encoded multiple virulence genes,and hypervirulent genes（iucABCD、iutA、rmpA、rmpA2 and iroN）were found in the virulence plasmids.（4）Analysis of genetic environment for resistance genes showed the complex resistance regions consist of resistance genes,insertion sequences and integrons.Class 1 integrons In191 and In27 were found in the pXJ-K1-p2 and pXJ-K2-p3,respectively.（5）Phylogenetic analysis showed that strains XJ-K1 and XJ-K2 closely genetic related.7.Esculetin has the best antibacterial activity in vitro against strains XJ-K1 and XJ-K2 among five traditional Chinese medicine monomers,and the MIC values of two strains were512μg/mL.8.The drug combination between esculetin and nitrofurantoin could reduce the MIC of nitrofurantoin against strains XJ-K1 and XJ-K2.The drug combination effect of esculetin with nitrofurantoin was synergistic（FICI<=0.5）against the extensively drug-resistant hypervirulent Klebsiella pneumoniae strain XJ-K1 and XJ-K2.Conclusion:1.Strains XJ-K1 and XJ-K2 were the ST11 extensively drug-resistant hypervirulent Klebsiella pneumoniae.2.A large number of resistance genes and virulence genes carried by strains XJ-K1 and XJ-K2 might the main reasons leading to extensively drug-resistant and hypervirulent.3.Esculetin has a certain antibacterial activity against extensively drug-resistant hypervirulent Klebsiella pneumoniae in vitro.The drug combination of esculetin with nitrofurantoin could increase the sensitivity of extensively drug-resistant hypervirulent Klebsiella pneumoniae against nitrofurantoin,which has synergistic effect.