Genomic Analysis And Interaction With Neutrophils Of Klebsiella Pneumoniae

Background:Hypervirulent Klebsiella pneumoniae(HvKP)is hypermucoviscous organism,carrying genes of rmpA and aerobactin,causing community-acquired serious infection and metastatical spread in young healthy hosts.HvKP is more virulent than classic Klebsiella pneumoniae(cKP).Neutrophils play an important role during innate immune response against some bacterial infection by phagocytosis and neutrophil extracellular traps(NETs).To investigate the mechanism of hypervirulence and pathogenicity of HvKP,Here,six HvKP strains were sequenced and analyzed by Third-generation sequencing technology,and the role of neutrophils in defending against HvKP was explored.Methods:To explore the genetic basis of pathogenicity and hypervirulence of HvKP,we selected and completed the genomes of six HvKP strains belonging to different serotypes(including two K1 HvKP strains,two K2 HvKP strains and two K57 HvKP strains),using PacBio single-molecular real-time(SMRT)technology,and conducted the study from virulence genes and resistance genes.Fourty-five Klebsiella pneumoniae strains were selected according to string test,capsular serotypes,rmpA and aerobactin detection,including HvKP-Kl strains(n=16),HvKP-K2 strains(n=14)and cKP strains(n=15).Neutrophils were purified from freshly drawn blood of healthy human volunteers.Neutrophil phagocytosis was assessed by flow cytometry at 0,10,30 and 60 min,and morphological changes of phagocytosed bacteria were examined by transmission electron microscopy at 30 and 60 min.The survival index was calculated to evaluate the intracellular killing by neutrophils at 60 min.Production and trapping of NETs were monitored by scanning electron microscopy and confirmed by immunostaining.Results:The six HvKP strains contained more virulence genes than cKP strains.The iucABCD gene encoding aerobactin and the iroBCDN gene encoding salmochelin present only in HvKP strains.The number of virulence genes carried by K1 HvKP strains was higher than that carried by K2 and K57 HvKP strains.There were 21 genes that present only in K1 HvKP,including 3 kfuABC genes encoding Kfu protein and 18 nitrogen acquisition genes.The 11420 and 11492 strains carried the carbapenem-resistant genes blaKPC-2 and ?-lactamase-resistant genes blaCTX-M-24,respectively.The results of phagocytosis assay showed that the rate of neutrophil phagocytosis against HvKP-K1,HvKP-K2 and cKP increased over time(0 min to 60 min).However,the phagocytosis rate of HvKP-K1 or HvKP-K2 was lower than that of cKP at three time stages[the phagocytosis rate of HvKP-Kl,HvKP-K2 and cKP was(28.36± 4.17)%,(30.46 ± 3.52)%and(45.89 ± 4.79)%at10 min;(39.13 ± 2.71)%,(41.96 ± 3.16)%and(57.39 ± 3.21)%at 30 min;(45.7±3.21)%,(47.24 ± 3.02)%and(60.29 ± 3.13)%at 60 min].The observation of transmission electron microscopy exhibited that neutrophils were able to ingest more cKP than HvKP-Kl.It was also observed that the bacterial cells of cKP displayed incomplete cell surface and seemed to be lysed,while the bacterial cell wall of HvKP-K1 remained intact and the cell division of HvKP-Kl was even visible.The survival index of HvKP-K1 and HvKP-K2[(1.0450± 0.1455)and(0.9820 ± 0.1013),respectively]was significantly higher than that of cKP(0.8038 ± 0.0876).Both HvKP-Kl and cKP were able to induce.neutrophils to release fiber-like NETs which were confirmed by immunofluorescence analysis,but NETs trapped more cKP than HvKP-Kl.Moreover,the trapped cKP showed pores on the surface,but this phenomenon didn't appear on HvKP-K1.Conclusion:HvKP carried more virulence genes than cKP,which explained the reason that HvKP possessed higher virulence.The killing by both intracellular and extracellular mechanisms of neutrophils to cKP was detected.HvKP was more resistant to neutrophil-mediated phagocytosis and intracellular killing than cKP.Although both HvKP and cKP induced NETs,more cKP than HvKP were trapped in NETs.