Genome Sequencing And Comparative Genome Analysis Of 6 Hypervirulent Klebsiella Pneumoniae Strains

Objective Hypervirulent Klebsiella pneumoniae(hvKP)has been increasingly reported in the Asian Pacific Rim and Western countries during the past three decades,which cause severe metastatic infections such as liver abscesses,endophthalmitis and meningitis.There are few studies on hv KP based on genomic background,which limits our understanding of hv KP from the whole genome level.Therefore,we performed a genome sequencing and comparative genome analysis of 6 hv KP with different capsular serotypes isolated in China aimed to increase our understanding of hvKP on genome level and to lay the foundation for exploring its pathogenic mechanism.Methods The string test(ST)was used to determine the hyperviscosity of six hvKP strains.The total genomic DNA of these strains were extracted and sequenced by an Illumina HiSeq 4000 sequencing platform.The data of the sequencing was processed and assembled using SOAPdenovo software.Glimmer software was used to predict genes from assembled result.The predicted genes were translated into amino acids and aligned to KEGG and COG databases for gene function annotation.Comparative analyses of genome with NTUH-K2044 showed the difference and evolutionary relationship between the sequencing samples and the reference sequence,including structural variation(synteny),gene family,unique genes,single nucleotide polymorphisms(SNPs)and small insertions and deletions(In Dels)and phylogenetic analysis.Finally,the VFDB database was used to predict the virulence genes in the genome and the virulence genes of Klebsiella pneumoniae were also searched in the literature.The selected virulence genes reference sequences were downloaded from the NCBI nucleic acid database,and made BLAST analysis between these reference sequences and whole-genome sequence of six hvKP strains,respectively.At the same time,SNP-based differentiation of K.pneumoniae virulence genes were also detected.Results The result of string test showed that all of six hv KP strains were hypermucoviscous strains.Genome sequencing and assembly results showed that the genome size of the six hvKP ranged from 5.34 Mb to 5.58 Mb,with 57.22%-57.46% GC content.Comparative analyses of genome with NTUH-K2044 revealed that the synteny in the structure showed insertions and deletions between the genomes and a lot of genome rearrangement events such as reversal,translocation and inversion;a large number SNPs and InDels were found compared with NTUH-K2044;phylogenetic tree based on full-genome SNP of the 7 hv KP showed that NTUH-K2044 formed a single clade,suggesting a distant evolutionary distances with other 6 strains,the five non-K1 hvKP strains have closer phylogenetic relationship;BLAST comparison analysis found that some selected virulence genes,such as kfu,allS,iucABCDiutA and so on,have different degrees of deletion in other five non-K1 hv KP.SNP-based virulence gene mutation analysis showed that some virulence genes such as mrkD,ureDABCEFG and other genes have different degrees of SNP mutations.Conclusion The whole genome sequencing of six hvKP strains and comparative genome analysis provide us with a basic understanding of the genome composition,genetic polymorphism,and evolution and virulence genes situation of the hvKP,which may lay the foundation for further research on gene function and pathogenesis of hvKP.