Clinical And Genomic Characteristics Of Multi-Drug Resistance Sequence Type 11 Klebsiella Pneumoniae During Inter-Host Transmission And Evolution

Objective:Sequence type(ST)11 Klebsiella pneumoniae is the main epidemic clone of Carbapenem-Resistant Klebsiella pneumoniae(CRKP)in China.The aim of this study is to firstly clarify the clinical and microbiological characteristics of ST11 Klebsiella pneumoniae,especially in clinical manifestations,prognosis,risk factors,antimicrobial resistance phenotype and virulence genotype.Then,we intend to clearly understand the genomic characteristics(resistance,virulence,and plasmid replication)and virulence phenotype of multi-drug resistance(MDR)ST11 Klebsiella pneumoniae during its transmission and evolution among different hosts.Finally,the target is to characterize the clinical and genomic feature of the MDR-ST11-hvKp,which is specially focus on the location of various key virulence genes and its associated genomic context in MDR-ST11-hvKp strains.It is essential to advance intervention and prevention of MDR-ST11-hvKp spreadMethods:(1)A retrospective study focused on 104 Kp infections isolated from March 2017 to June 2018 was conducted.Various clinical data were collected.We detected the capsule serotype(K1,K2,K5,K20,K54,K57)and key virulence genes(peg-344,peg-589,rmpA,rmpA2,iucA,iroB)by the Polymerase Chain Reaction.Additionally,the string test was performed to confirm the hypermucoviscosity phenotype.The combination of rmpA,rmpA2,iucA,iroB,and peg-344 was defined as hypervirulent Klebsiella pneumoniae(hvKp).A case-control study was conducted to determine the clinical and microbiological(virulence genotype,antimicrobial resistance and virulence phenotype)characteristics of ST11 Klebsiella pneumoniae.(2)Next-genome sequencing and various virulence phenotype tests were conducted within the ST 11 Klebsiella pneumoniae strains.Relying on various databases,liking ResFinder,Virulence Factor Database,and PlasmidFinder,the antimicrobial resistance genes,virulence genes,and plasmid replication were analysised.Refering HS11286(ST11 Klebsiella pneumoniae)sequencing data,the phylogenetic tree of all the ST11 Klebsiella pneumoniae strains was constructed to clearly understand their evolutionary relationship.The virulence phenotype was evaluated in Galleria mellonella model.Moreover,the biofilm formation and serum killing experiment were also assessed Combined different single nucleotide polymorphisms(SNPs)and clinical information,the transmission and evolution rountine of MDR ST11 strain was mapped.(3)Based on the previous hvKp definition,the clinical characteristics of the MDR-ST11-hvKp were analysed by using a case-control method.Twelve MDR-ST11-hvKp strains were selected to be sequenced by the Nanopore.Then,we combined next-generation sequencing data with single-molecule sequencing technology to map a distinct genome.The BRIG software was used to analyze the chromosomes of MDR-ST11-hvKp strains and its virulence-associated plasmids,respectively.Previously published ST11 carbapenem-resistant hvKp(CR-hvKp)genomes were used as reference to clarify the genetic relationship between MDR-ST11-hvKp isolated from our hospital and ST11 CR-hvKp that induced fatal infections in Zhejiang province.The location of key virulence genes and its genomic context were ultimately confirmed among different sub-clones of MDR-ST11-hvKp for accurate prevention and control of the "superbug" infection.Result:(1)Among 104 patients infected with Klebsiella pneumoniae,the average age was 65.65 ± 17.99 years old and 62 cases(59.6%)were males.The main prevalent sequence type in this study is ST11,which accounts for 40 cases.Sixty-five cases(62.5%)Were defined as hvKp strains.In the ST11 group,35 cases(87.5%)were hospital-acquired infections,and 27 cases(67.5%)were from ICU.A significantly higher incidence of pneumonia(P=0.017)was caused by ST 11 group,but only two(5.0%)cases induced bloodstream infection.92.5%(37 cases)were under catheterization status and 97.5%(39 cases)patients had a history of various antimicrobial agents exposure within 90 days.In the ST11 group,the incidence of poor prognosis at 30 days reached 42.5%,which was significantly higher than that of the control group.Multivariate logistic analysis showed that catheterization status(OR,6.094)and hospitalization>2 days within 90 days(OR,5.517)were independent risk factors for ST 11 infection.All ST 11 strains showed multi-drug resistant phenotype(MDR).Moreover,the resistance rates to imipenem and meropenem were 87.5%and only 5(12.5%)strains were resistant to tigecycline.None of the strains harbored Kl,K2,K5,K20,K54 and K57 capsular serotype.Fifteen strains(37.5%)presented hypermucoviscosity phenotype and seventeen isolates(42.5%)were defined as MDR-ST11-hvKp strains.The dominant virulence genotype was iucA+rmpA2(13/40,32.5%).(2)Next-generation sequencing and virulence phenotype were performed in thirty-three MDR ST11 Klebsiella pneumoniae,which contains thirteen MDR-ST11-hvKp strains.Of the thirteen MDR-ST11-hvKp strains,84.6%(I1/13)were defined as hospital-acquired infections and 2 cases(15.4%)were community-acquired infections.All the MDR-ST11-hvKp strains could form biofilms but were negative in serum killing tests.There exist two unique virulence genotypes within the MDR-ST11-hvKp group:eleven strains carried rmpA2+iucA,and two strains presented peg-344+rmpA+rmpA2+iucA.Interestingly,only eight(61.5%,8/13)hvKp isolates expressed virulence phenotype in Galleria mellonela model.All MDR-ST11 Kp carried fosA resistance genes,31 strains(31/33,93.9%)harbored blakPC-2 resistance genes and 21 strains had tetA resistance genes In addition,rmtB and blaTEM were co-detected in 25 strains.A total of 1313 high-quality SNPs(hqSNPs)were found.Based on the 749 hqSNPs,these MDR ST11 strains could be divided into two Clades(totally 9 clonal subtypes),which contains 372 unique hqSNPs Clade 1 and 377 unique hqSNPs Clade 2.There exited six strains in Clade 1,five of which were hvKp.Clade 2 includes 27 strains,which were divided into two clusters:Clade 2a(contains 5 hvKp)and Clade 2b(contains 3 hvKp).Of the nine ST11 subclones,six subclones were belonged to hvKp.Additionally,all the ST 11 strains contained the IncFII plasmid replicon and more than three plasmid replication types were presented in 81.8%(27/33)strains,suggesting that the ST11 group carried various plasmids.Interestingly,hvKp in the Clade 1 branch carried the IncHI1B plasmid replicon,whereas none of the hvKp strains in Clade 2 carried the IncHI1B,suggesting that MDR ST11 group acquired key virulence-associated genes/plasmids during transmission and evolution among different hosts and then confered into MDR-ST11-hvKp.(3)Multivariate logistic analysis showed that cerebrovascular disease(OR,4.706)and metastatic infection(OR,3.495)were independent risk factors for MDR-ST11-hvKp infection.Twelve MDR-ST11-hvKp strains were selected for single-molecule sequencing.It is noted that three hvKp of the Clade 1 carried pVir-CR-hvKp4-like plasmid and the other two hvKp strains of the Clade 1 contained the pLVPK-like virulence plasmid.The virulence-associated plasmid of four hvKp strains from Clade 2a were similar to the WCHKP020030 plasmid pOXA1₀20030,but the virulence-associated in the Z29 strain were partially similar to the WCHKP020030 plasmid pOXA1₀20030,suggesting that a novel plasmid harbored key virulence genes is emerging.Most importantly,the key virulence genes of the hvKp in Clade 2b located in the chromosomeConclusion:(1)HvKp may be replacing classical Klebsiella pneumoniae as the dominant sub-pathotype in our hospital and it is alarming that MDR-ST11-hvKp emerged.The main infection type caused by ST 11 Klebsiella pneumoniae was hospital-acquired infection,especially in ICU and the prognosis is poor in 30 day.The independent risk factors for ST11 infection are hospitalization>2 days within 90 days and catheterization status.(2)During the evolution and transmission among different hosts,MDR ST11 Klebsiella pneumoniae acquired different key virulence gene/clusters and evolved into multiple sub clonals However,heterogeneity of virulence phenotype was shown in Galleria mellonella model MDR-ST11-hvKp strain with strong biofilm formation emerged in our hospital and might have transmitted into the community.(3)The independent risk factors for MDR-ST11-hvKp infection are cerebrovascular disease and metastatic infection.The key virulence gene cluster iucA+rmpA2 previously located in virulence plasmid could insert into various chromosomes and antimicrobial resistant plasmids among different ST11 sub clones during inter-hosts evolution and transmission,suggesting that the stable MDR-ST11-hvKp is emerging and the genomic context of key virulence gene/clusters is diversity.