Clinical Infection Characteristics,Molecular Typing And The Differences Of Fitness After Acquisition Of Carbapenem-Resistance Gene Of Hypervirulent Klebsiella Pneumoniae By Bloodstream Infection

Objective:To investigate the clinical infection characteristics,drug resistance status and molecular biological features of bloodstream infection Klebsiella pneumoniae（KPN）.To analyze the biological differences and virulence changes of ST23 type hypervirulent K.pneumoniae（hv KP）after acquiring KPC and NDM type carbapenem resistance genes,respectively,and to preliminarily explore the fitness characteristics and differences in gene expression levels of the constructed isolates of carbapenem-resistant hv KP（CR-hv KP）.Methods:（1）Non-replicated KPN isolates and clinical data were collected from patients with bloodstream infections from 06/2019 to 10/2022,excluding incomplete medical history data and mixed infections with two or more bacteria,and finally a total of 240 KPN isolates were included in this study.The Microflex LT mass spectrometer was used for isolates identification,and the Vitek-2 analyzer was used for antibiotic sensitivity testing.KPN were divided into hv KP and common KPN（c KP）groups by virulence gene type.The current status of resistance in bloodstream infected KPN and major resistance gene carriage were analyzed.Multi-locus sequence typing（MLST）and podoconjugate serotyping（wzi）were applied to analyze the affinity of each ST type of KPN.（2）ST23^K1hv KP carrying the p LVPK-like virulence plasmid was selected as the parental generation（KP54798）,and its whole genome was sequenced to analyze the plasmid carriage status,and confirmed as ST23^K1type and carrying the p LVPK-like plasmid.The recombinant resistance plasmids p ACYC-184^KPC(p A^KPC)and p ACYC-184^NDM(p A^NDM)were introduced into parental generation to construct CR-hv KPs(KP54798-p A^KPCand KP54798-p A^NDM).The biological characteristics of parental KP54798,constructs KP54798-p A^KPCand KP54798-p A^NDMwere analyzed using antibiotic sensitivity testing,String test,biofilm formation assay and Capsular polysaccharide serotyping quantification assay.The cost of finess was compared by co-culture competition assay,growth kinetic curve and plasmid stability assay.The serum bactericidal assay and the Galleria mellonella infection model were used to compare the virulence changes of the three,which were analyzed at the gene transcriptome level using RNA transcriptome sequencing.Results:（1）A total of 240 KPN isolates were included in this study,hv KP had an overall detection rate of 44.2%,predominantly community infections,accounting for 64.2%,with a high prevalence in young adults between 30 and 60 years of age,accounting for 53.8%;mainly distributed in infection units（30.2%）and intensive care units（14.2%）.c KP had a detection rate of 55.8%,c KP was predominantly hospital-acquired,accounting for 76.9%,mainly concentrated in elderly people over 60 years old,accounting for 56.7%;the main clinical distribution was in intensive care units（14.2%）and emergency departments（14.2%）.The antibiotic sensitivity testing results showed that the resistance rates of hv KP and c KP to carbapenems were 13.2%and 20.1%,respectively,among them,ST23 type（38.7%）was predominantly detected in hv KP.Goe BURST analysis showed that ST23 was the main clonal group type in hv KP group,and K1 type was predominant in wzi typing;c KP ST11（20.1%）was the predominant type in c KP,and K64（19.4%）was the predominant wzi typing,CR-hv KP was divided into ST11（78.6%）and ST23（21.4%）.Analysis of common resistance genes:the detection rates of five drug resistance genes bla _KPC,bla _NDM,bla _IMP,bla _OXA-48and bla _VIMin c KP were 16.4%,4.5%,1.5%,0.7%and 0,respectively;in hv KP,only the resistance genes bla _KPC（11.3%）and bla _NDM（0.9%）were detected,respectively accounting for 92.3%and 7.7%of CR-hv KP,respectively.For virulence genes,the detection rates of hv KP were all higher than 75%.The detection rate of c KP group was lower than 40%except for mrk D and kfu.（2）The genome of clinical isolate KP54798 consisted of a chromosome with a genome length of 5420844 bp and a plasmid with a molecular weight size of 225890 bp.Phenotype validation determined that the CR-hv KP isolates were successfully constructed and that the constructs were significantly more resistant to drugs.After antibiotic stress screening for carbapenem-resistant mutants,it was found that the KPC-positive isolate carried the exogenous plasmid stably with a carriage rate of 92.3±0.58%after 72 h of continuous transmission,while the NDM-positive strain carried only 42.0±2.65%after 72 h of continuous transmission.The constructs CR-hv KP showed different degrees of reduction in growth rate,biofilm formation ability and virulence.Whole-transcriptome sequencing showed that the functional genes down-regulated in the KPC-positive isolates were involved in podoconjugate metabolism,while the NDM isolates showed no significant down-regulation,while the up-regulated functional genes were concentrated in biosynthesis.Compared with the parental generation,the KPC-positive isolates showed down-regulated expression levels of virulence genes rmp A,rmp A2,iuc A and iro B,down-regulated expression levels of podosomal polysaccharide structure genes man C,wza and wzb,and down-regulated expression levels of related regulatory genes Rcs A/B（P<0.05）.Conclusions:（1）The overall detection rate of our hv KP was 44.2%,with ST23^K1type predominating,and the detection rate of virulence genes was higher,and ST23^K1type CR-hv KP was detected.（2）KPC type CR-hv KP carried stable exogenous plasmids,but showed obvious fitness changes such as weakened high viscosity phenotype and reduced virulence,while NDM type CR-hv KP high viscosity phenotype and virulence were slightly reduced,but plasmid carriage was unstable.KPC-type CR-hv KP showed a decreasing trend in the expression levels of major virulence genes,podosome polysaccharide structure and related regulatory genes,so its fitness cost characterization may be related to the down-regulation of the above genes expression.Meanwhile,the overall gene expression of the construct CR-hv KPs were altered after the introduction of the exogenous plasmid resulting in changes in its physiological characteristics.