Molecular Epidemiological Analysis Of CR-hvKP Carbapenemaseresistant OXA-48-like And Exploration Of Virulence And Resistance Mechanisms

Objective:To study the clinical distribution and molecular epidemiological characteristics of carbapenem-resistant and highly virulent Klebsiella pneumoniae in our hospital;to study the clinical distribution and molecular epidemiological characteristics of drug resistance and virulence of bla _OXA-48-likepositive CR-hv KP strains.Method:1.The carbapenem（ertapenem,meropenem,or imipenem）-insensitive Klebsiella pneumoniae isolates（CRKP）isolated from various types of clinical specimens from inpatients at the First Affiliated Hospital of Nanchang University from April 2020 to March 2022 were collected as test strains,and duplicate strains isolated from the same patient were excluded.Species identification of all strains was performed by the VITEK 2 automated system,and basic clinical information was collected from patients using the hospital inpatient system.The carbapenemase resistance genes bla_KPC、bla_NDM、bla_OXA-48、bla_IMP、bla_VIM,and p LVPK virulence plasmid-associated genes rmp A,rmp A2,iuc A,iro B,peg-344 were detected by PCR,and any one or more of these virulence genes were defined as carbapenem-resistant high virulence.Klebsiella pneumoniae（CR-hv KP）,and data were presented and statistically analyzed using Graphpad prism 9.0 and SPSS26.0 software.2.CR-hvKP strains carrying both the p LVPK virulence plasmid and the bla _OXA-48-likegene,defined as bla _OXA-48-likeCR-hv KP,were screened for strain identification and drug sensitivity testing using the Vitek 2 Compact fully automated bacterial identification and analysis system.PCR was performed to detect the capsular serotype genes（K1,K2,K5,K20,K54,K57,K64）,β-lactamase-encoding genes(bla _CTX-M,bla _SHV,bla _TEM)and plasmid replicon typing of bla _OXA-48-likeCR-hv KP strains.Homology analysis of bla _OXA-48-likeCR-hv KP was performed using multilocus sequence typing（MLST）and pulsed-field gel electrophoresis（PFGE）.The virulence phenotype of bla _OXA-48-likeCR-hv KP was analyzed in combination with the string test,Gallonella infection type test,serum resistance test,and biofilm formation test in vitro,the plasmid location of the virulence plasmid（rmp A2）was determined by S1-PFGE and Southern blot,and the transferability of carbapenemase resistance plasmid was detected by conjugation experiments.Results:1.A total of 217 CRKP strains were collected from April 2020 to March 2022 in this hospital.The detection rates of CRKP virulence factors peg-344,rmp A,rmp A2,iuc A,and iro B were 16.13%,19.35%,73.27%,64.98%,and 24.42%,respectively.Carbapenem resistance genes were mainly bla _KPC（92.17%）,followed by bla _OXA-48（23.04%）and bla _NDM（11.98%）.bla _IMPand bla _VIMgenes were not detected.The detection rate of CR-hv KP was 83.87%,and the detection rate of rmp A2 gene was>80%.Almost all CR-hv KP strains carried one or more drug resistance genes.The mean age of patients with CR-hv KP infection was 55.91±15.37 years,the ratio of male to female infection was 2.31,and the overall mortality rate was 43.41%（79/182）.The distribution of infected patients was mainly in neurosurgery（21.43%）,emergency medicine（15.93%）,and critical care medicine（13.19%）.Clinical specimens of the strain were mainly isolated from sputum（37.33%）,and the differences were statistically significant（P<0.05）between patients with CR-hv KP infection who improved and those who died in terms of six factors,including length of stay≥30 days,history of ICU admission during hospitalization,history of recent surgery,comorbid diabetes mellitus,cardiovascular disease,and exposure to≥2 types of antibiotics before infection.2.A total of 50 strains of bla _OXA-48-likeCR-hv KP were detected,with a detection rate of 27.47%.The common bla _OXA-48-likecarbapenemase resistance genes were bla _OXA-181and bla _OXA-232,both carrying at least one carbapenemase gene(bla _KPC,bla _NDM,bla _OXA-48)or broad-spectrumβ-lactamase genes(bla _CTX-M,bla _SHV,bla _TEM).The PBRT of bla _OXA-48-likeCR-hv KP was dominated by Col E-type plasmids,Inc F-type plasmids and Inc X3-type plasmids,with Inc F+col E as the predominant replicon typing combination.All strains showed very high resistance to clinically used antimicrobial drugs.MLST and PFGE suggested the existence of both clonal and plasmid-borne genetic relationships.bla _OXA-48-likeCR-hv KP was mainly clustered in K64 ST11 type and K47 ST15 type,and virulence phenotyping experiments suggested that bla _OXA-48-likeCR-hv KP virulence The S1-PFGE results indicated that the bla _OXA-48-likeCR-hv KP plasmid profiles showed diversity.Eight strains carrying three carbapenem resistance genes(bla _KPC,bla _OXA-181/_OXA-232and bla _NDM)CR-hv KP were isolated for the first time,and Southern blot suggested that all carried p LVPK-like virulence plasmids,and all eight strains were successfully conjugated into EC600 strains.Conclusion:1.CR-hvKP showed a high detection rate in this institution.The main resistance mechanism of CR-hv KP strains was the carrying of bla _KPC、bla _OXA-48and bla _NDMgenes and the co-existence of multiple resistance genes.2.The most common OXA-48-like carbapenemase resistance genes are bla _OXA-181and bla _OXA-232,mainly clustered in strains K64 ST11 and K47 ST15,which have been validated as highly virulent strains in both in vivo and in vitro virulence tests,and we should actively detect and take preventive and control measures against these bacteria.3.First report of co-production of three carbapenemase genes(bla _KPC+bla _NDM+bla _OXA-181/bla _OXA-232)and p LVPK-like virulence plasmids in CR-hv KP isolates.