Molecular Mechanism Of PLVPK-like Virulence Plasmid Mutation And Evolution In Carbapenem-resistant Klebsiella Pneumoniae (CRKP)

The pLVPK plasmid,discovered in Taiwan in the hypervirulent Klebsiella pneumoniae（hv KP）,can mediate the enhancement of the virulence of the strain.It is a typical sign of the hv KP circulating in the community.While,the carbapenem-resistant Klebsiella pneumoniae（CRKP）epidemic in the hospital have a broad drug resistance spectruma and low virulence.The MDR strain represented by ST11 CRKP reported in The Lancet Infectious Diseases in 2018 has a broadly resistant and hypervirulent due to the pLVPK-like plasmid,which intensifies the clinical practice of CRKP.The pLVPK virulence plasmid was originally reported to carry neither drug resistance gene nor transferability.Later,it was discovered that the virulence plasmid carrying the drug resistance gene and the integration of the virulence plasmid fragment on the drug resistance plasmid showed that the virulence plasmid was in Evolutionary recombination has occurred in CRKP.This study started with a ST15 type CRKP,with widely drug-resistant and virulence factors,explored the evolutionary characteristics and transmission laws of virulence plasmids in CRKP.It provides a theoretical basis for the prevention and control of the broadly drug-resistant and hypervirulent strains caused by the further evolution and spread of virulence plasmids in CRKP.This study found that 2 CRKP（KP17-15 and KP17-16）isolated from a hospital in Pingdingshan City,Henan Province have the characteristics of extensive drug resistance.The results of drug susceptibility experiments showed that the two CRKP strains had the same drug resistance spectrum,and were resistant to almost all drugs but fosfomycin against Enterobacter infections,including tigecycline and polymyxin.PCR detection revealed that the 2 CRKP simultaneously carried the virulence factor rmpA₂ and iucABCD-iutA gene clusters that existed on the virulence plasmid.Based on the Illumina and Nanopore whole-genome sequencing,it was found that two ST15 CRKP strains with XDR phenotype isolated from the same ward had almost the same genomic characteristics and belonged to the same clone.The conjugable hybrid virulence plasmid p17-15-vir（～479 kb）isolated from KP17-15 simultaneously carries 17 drug resistance genes,virulence factors rmpA₂,iucABCD-iutA,and the complete conjugative transfer functional region tra-trb gene cluster.Comparative analysis found that the plasmid might be formed by recombination of the virulence plasmid p17-16-vir（～290 kb）and the conjugative resistance plasmid p17-16-CTX（～188 kb）isolated from KP17-16 under the two homologous regions encoding group II intron reverse transcriptase and mobile element ISShes11.In addition,p17-16-vir and p17-16-CTX can undergo fusion recombination during the conjugative transfer process to form a p17-15-vir-like plasmid,but the recombination is different from p17-15-vir.On the one hand,This discovery confirms our speculation on the formation of the hybrid plasmid carried by KP17-15.On the other hand,the recombinant evolution of drug-resistant plasmid and virulence plasmid in CRKP will lead to the generation of conjugable plasmid carrying both virulence and resistance factors.At the same time,we found that the non-conjugating virulence plasmid p17-16-vir can undergo horizontal transfer with the help of the conjugating plasmid.We found that a CRKP strain KP19-2029 isolated from a blood specimen from a teaching hospital of Zhengzhou University carries both rmpA₂ and iucABCD-iutA gene clusters,and has a phenotype with enhanced virulence（the median lethal dose of mice is 1.0×10⁵ CFU）.Using rmpA₂ probe to perform Southern hybridization on the genome isolated by S1-PFGE,no pLVPK-like virulence plasmid was detected.Whole-genome sequencing analysis revealed that a～104 kb fragment derived from the p17-15-vir plasmid containing rmpA₂ and iucABCD-iutA gene cluster was integrated into the cbp gene cluster of the bacterial genome mediated by IS26elements.In order to verify whether the virulence of the strain is increased due to the insertion of this fragment,we used CRISPR/Cas9 technology to knock out rmpA₂,which plays an important role in regulating virulence.But there are no difference in the virulence between KP19-2029 and KP19-2029△rmpA₂.There may be other mechanisms that mediate the enhancement of the virulence of the strain.However,the integration of virulence plasmid fragments into the genome will be more stable,providing stable and convenient conditions for the horizontal spread of virulence gene clusters while accompanying the vertical spread of the genome.In summary,we found that the pLVPK-like virulence plasmid has further evolved in CRKP,which is shown as follows:1.The virulence plasmid can fuse with the conjugative drug-resistant plasmid to form a hybrid conjugative plasmid that carries both drug resistance and virulence factors.And a horizontal transfer occurs.2.The non-conjugating pLVPK-like plasmid can be transferred horizontally with the help of the conjugating plasmid;3.The virulence plasmid fragment carrying the virulence factor can be integrated into the CRKP genome through the IS transfer element and accompany the genome vertically spread.The above findings enrich the evolutionary theory of pLVPK-like plasmids and provide a theoretical basis for the control of this type of plasmids.