Resistant Mechanisms And Molecular Epidemiology Of Carbapenem-resistant Serratia Marcescens Strains From Patients In Neurosurgical ICU

Objective: To investigate the molecular mechanisms and epidemiology of carbapenem-resistant Serratia marcescens(S.marcescens)strains from patients in intensive care unit(ICU)of neurosurgery through detecting the resistant phenotype,genotype and homology of these strains,as well as the transfer charctersitic and biological information of the plasmids in these strains.The aim of this study is to provide theoretical basis for the antibiotic therapy and the prevention and control of nosocomial infection.Methods:(1)Six carbapenemases resistant S.marcescens strains isolated from sputum samples from six patients in the neurosurgical ICU in a hospital of Qingdao from Oct.2014 to Mar 2015 were selected for identification and susceptibility testing using Vitek-2 Compact automatic microbiological analysis system.The modified Hodge test(MHT)was used to detect carbapenemase.(2)Polymerase chain reaction(PCR)and multiplex PCR were used to detect whether carbapenem,extended-spectrum beta lactamase(ESBL)and cephalosporinase(Amp C)coding genes and quinolone,aminoglycoside and fosfomycin resistance genes were carried.The positive genes were confirmed by using PCR and product sequencing.(3)The homology between strains was detected by pulsed field gel electrophoresis(PFGE).(4)The transfer charctersitic of the plasmid was analyzed by conjugation assay.(5)Bacterial genomic DNA was sequenced by the second-generation Mi Seq sequencing platform,and the resistance genes,mobile elements and other features of the plasmid were annotated by Res Finder,The Transposon Registr,ISfinder and INTEGRALL online databases.Gene organization diagrams were drawn in Inkscape0.48.1.Results:(1)Phenotypic profiles of 6 S.marcescens strains were consistent and were resistant to ceftriaxone,aztreonam,ertapenem,imipenem and meropenem as well as fosfomycin.All the six strains were positive in MHT.(2)Gennotypic profiles of 6 S.marcescens strains were also consistent and all carried four resistance genes: carbapenemase coding gene bla KPC-2,ESBLs coding gene bla SRT-1,aminoglycosamine resistance gene aac(6')-Ic and fosamycin resistant gene fos A7.(3)The PFGE bands of six S.marcescens strains were identical.According to Tenover rule,the six S.marcescens strains were clonally related and belong to the clonal group(inditinguishable),that is,outbreak strain.(4)The plasmid carrying bla KPC-2 gene was successfully transferred to E.coli J53 AziR.S1 nuclease digestion and PFGE showed that both the donor bacteria and their transconjugants carried a plasmid band about 100 kb in size.The transconjugants were all resistant to ceftriaxone,aztreonam and ertapenem,intermediate to imipenem and meropenem,and sensitive to other drugs.Only bla KPC-2 gene was detected in the transconjugants,but not bla SRT-1,aac(6')-Ic and fos A7 genes.(5)Bioinformatics analysis showed that a novel plasmid,named as p S1-KPC2(Gen Bank acc number: MN615880),was found in S.marcescens isolates.The plasmid belonged to the incompatibility group of Inc FIIK and contained bla KPC-2 gene which was encoded by transposon?Tn6296.Conclusion:(1)The carbapenem-resistant S.marcescens strains shows multiresistant phenotype and genotype,and production carbapenemase,which is encoded by bla KPC-2 gene,is the mechanism of resistance to carbapenem antibiotics.(2)The cloning spread of S.marcescens strains caryring bla KPC-2,bla SRT-1,aac(6')-Ic and fos A7 resistance genes is the cause of the outbreak of nosocomial infection in patients in ICU of neurosurgery department,which is the first report worldwilde,to our knowledge.(3)The transfer charctersitic of the Inc FIIK plasmid and the structural characteristics of transposon ?Tn6296 facilitate the horizontal transmission of blaKPC-2 gene.