Molecular Epidemiology,Drug Resistance And Pathogenic Mechanism Of Tigecycline-non-susceptible Klebsiella Pneumoniae Was Researched In A Jiang Xi Tertiary Care Hospital

Objectives:1.To study the molecular epidemiological characteristics of tigecycline-non-susceptible Klebsiella pneumoniae.2.To research the genomics and mechanisms of drug resistance and virulence in tigecycline-non-susceptible Klebsiella pneumoniae.3.Explored the complete genome map and virulence characteristics of highly virulent tigecycline-non-susceptible Klebsiella pneumoniae（strain No.25）.Methods:1.A total of 5,729 non-repeated Klebsiella pneumoniae strains isolated from various clinical specimens in the Hospital from 2017 to 2020 were collected,and 34tigecycline-non-susceptible Klebsiella pneumoniae strains were screened by the microdilution method.Obtain clinical information of strains and distribution of specimen types by consulting medical records.Molecular epidemiological characteristics such as drug resistance genes and virulence genes were studied by PCR and sequencing.The genetic relationship of the strains was analyzed by PFGE and MLST.2.The second-generation sequencing technology was applied to 34tigecycline-non-susceptible Klebsiella pneumoniae strains to further understand their biological characteristics at the genome level,including virulence genes,drug resistance mechanisms,plasmid typing,and evolutionary relationships between strains.The possible drug resistance mechanism of tigecycline-insensitive Klebsiella pneumoniae was analyzed from the perspective of genomics.3.For a highly virulent tigecycline-non-susceptible Klebsiella pneumoniae strain No.25 screened in the first and second parts,the third-generation sequencing technology was used to draw a complete genome map,and its virulence plasmids and virulence plasmids were analyzed.Genomic characterization of drug-resistant plasmids.The genome of the virulence large plasmid of this strain was compared with4 known classical virulence plasmids.Finally,the virulence of strain 25 was verified by the combination of biofilm test,serum resistance test,invasion test of G.mellonella and mouse killing test.Results:1.The 34 strains of TNSKP screened showed low sensitivity to all drugs,with the exception of ertapenem,imipenem,meropenem and amikacin,the resistance rates were lower than 90%,and the other were higher than 90%or even reached 100%.Epidemiological analysis showed that most strains（55.9%）originated from intensive care units.The average age of the patients was older（57.19±21.65 years）,the length of hospital stay was long（42.5±30.1 days）,and most of them were in the state of intubation（76.5%）.The molecular epidemiological results of 34 strains indicated that PFGE could be divided into 6 genotypes,28 strains（82.4%）of type A were the main cluster,and most of them were ST11 at the same time;the capsular serotype was K14.K64.main.Drug resistance gene detection showed that 30 carbapenem-resistant strains all carried bla _KPC-2 gene,and 29 strains carried bla _SHV-12,bla _TEM-1,bla _CTX-M-65and bla LAP-2β-lactamase genes at the same time.The detection of tigecycline resistance genes showed that tet A and rps J genes were detected in other strains except strain 44,but no mutation was found.The virulence gene test found that some strains carried rmp A,rmp A2,uge,mrk D,kpn and other virulence genes,and all strains could detect mrk D gene carrying,so 29 strains carried mrk D,kpn and uge at the same time.3 strains carried both mrk D and kpn virulence genes,and only one strain,No.25,carried five virulence genes,rmp A,rmp A2,uge,mrk D,and kpn.2.Whole-genome analysis of all TNSKPs by second-generation sequencing technology showed that the multidrug-resistant efflux proteins Acr B,Mdt K and Mdt G could be annotated in all strains,and all strains were also found to carry acr A and acr B resistance in comparative genome analysis Gene.After comparing the virulence factor database,it was found that the virulence factors of the TNSKP strain in this study were mainly related to the siderophore,adhesion and biofilm formation of bacteria.In addition,annotated by plasma finder,it was found that the plasmid replicons were mainly of Inc R+Inc FII+Col RNAI type（91.2%）,and a strain carrying the same virulence gene cluster（iuc A+rmp A）as the p Vir-CR-hv Kp4 plasmid was found（strain No.25）.3.A highly virulent tigecycline-non-susceptible Klebsiella pneumoniae（TNS-hv Kp）was screened from the first two parts,and the complete genome of strain No.25 was obtained by combining the second and third generation sequencing technologies.First,through the annotation of drug resistance genes and virulence genes in the database,it was found that the plasmid of strain 25（plasmid 3）carried drug resistance that mediates resistance to carbapenems,β-lactams,quinolones and tetracyclines.Gene,plasma 1 carries the virulence gene cluster of iuc ABCD+iut A+rmp A.The genome of this virulence plasmid and four classical virulence plasmids were compared and found to have certain similarity.Through biofilm formation test,serum resistance test,invasion test of G.mellonella and mouse killing test,it was found that its virulence is no less than that of Klebsiella pneumoniae NTUH-K2044.Conclusions:1.TNSKP in this hospital mainly produces bla_kpc-2,the capsule serotype is mainly K14.K64,and ST11 is the main popular clone.2.The next-generation sequencing technology revealed that the multidrug-resistant efflux pump proteins Acr B,Mdt K and Mdt G,as well as acr A and acr B resistance genes were closely related to tigecycline insensitivity.3.Strain 25 is a tigecycline-non-susceptible Klebsiella pneumoniae that carries a virulence gene cluster（iuc ABCD+iut A+rmp A）on a plasmid and has multiple resistance genes,shows a high virulence phenotype.