Study On Resistant Mechanisms And Molecular Epidemiology Of Carbapenemase-producing Enterobacteriaceae

The emergence of carbapenem-resistant Enterobacteriaceae(CRE)has become a challenge for clinical therapy.We desigened this study to realize the sensitive rate changes of Klebsiella pneumoniae and Escherichia coli from 2011-2015 and investigate the main carbapenemase genes and the prevalence of carbepenem-resistant Enterobacteriaceae(CRE).We collected the carbapenemase-producing E.coli for futher research to estimate if the outbreak existed in Ruijin hospital.And some uncommon carbapenemases was studied to better understand their resistance characteristics and transmission mechanisms.This work will offer the guidance forclinical medication and control measures.Four parts were contained.Part 1: Sreening of carbapenemase-producing CRE in Ruijin hospital All the drug sensitivity test of K.pneumoniae and E.coli from 2001 to 2015 were collected from Department of Clinical Microbiology of Ruijin hospital,and analyzed by WHONET 5.6.Since 2009,K.pneumoniae showed a sustained decline of sensitivity rates to kinds of antibiotics.E.coli exhibited high sensibility to carbapenem,but drop rapidly to cephalosporins.One hundred and ninety-nine carbapenem-resistant Enterobacteriaceae(CRE)were obtained from Ruijin hospital from April 2010 to January 2015.The occurrence of blaKPC,blaIMP,blaVIM,blaOXA-48,blaNDM,blaSME and blaIMI genes was screened by PCR amplification and sequencing.A total of 124 strains were turned out producing carbapenemases.Among them,K.pneumoniae(60 isolates)and E.coli(39 isolates)were the most commom,and 88 KPC-2-,19 IMP-,15 NDM-and 2 IMI-producing isolates included.Part 2: Resistant mechanisms and molecular epidemiology of carbapenemase-producing E.coli Among the 21 E.coli stains isolated from 2011 to 2014,we identified 13 KPC-2-and 3 IMP-4-producing strains.All carbapenemase-producing E.coli strains were multidrug resistant.ST131 was the predominant sequence type(11 isolates),and 9 of them were H30 subclone.Nine of the 16 isolates were clonally related and their PFGE patterns were designated as A.Ten isolates transferred resistant plasmids to E.coli J53.Most blaKPC-2 genes were located on Inc FII plasmids ranging from 55 kb to 170 kb,while the blaIMP-4 genes were all carried on 50 kb Inc N plasmids.Part 3: Detection and molecular epidemiology of blaNDM-5-producing Enterobacteriaceae Four NDM-5-producing strains were idengified,including 1 K.pneumoniae,1 Proteus mirabilis and 2 E.coli.All K.pneumoniae and E.coli isolates were mutidrug resistance but susceptible to amikacin.Two E.coli isolates were ST453 and ST156 respectively and no correlation has found according to PFGE patterns.A novel sequence type of the K.pneumoniae was identified,ST2250.In 2 isolates,blaNDM-5 was located on conjugative Inc X3 plasmids with nearly the same size(~40 kb).4 isolates shared the same genetic content: IS3000,ISAba125Δ,IS5,blaNDM-5,bleMBL,trp F,dsb C,cut A1Δ,IS26 and umu DΔ.Part 4: Detection and molecular epidemiology of blaIMI-producing Enterobacteriaceae Both the 2 IMI-producing isolates were resistant to carbapenem antibiotics but were sensitive to expanded-spectrum cephalosporins,amikacin and ciprofloxacin;in both cases,the resistant blaIMI gene was located on plasmids.In Raoultella ornithinolytica isolate,blaIMI-3 was carried by plasmid p RJ46 C,which was identified to be a 166,620-bp conjugative Inc FIIY plasmid.It contained 193 ORFs with an average GC content of 52.8%.The blaIMI-3 gene was located on a novel transposon,Tn6306.Tn6306 was 15-kb in size,flanked by ISEcl1-like elements and 9-bp target site duplications.The blaIMI-2-carrying Inc FIB plasmid(p RJ18)in E.coli had a similar size to p RJ46 C.In summary,K.pneumoniae and E.coli isolates were the major components of CRE in Shanghai.The production of carbapenemases was one of the main causes of carbapenem resistance,with KPC-2,IMP and NDM most popular here.Both the horizontal transfer and clone spread mediate the dissemination of carbapenemase-producing E.coli in Ruijin hospital.E.coli ST131 subclone H30 may play an important role in the dissemination of KPC-2.blaNDM-5 was first identified in P.mirabilis,implying its further spread to Enterobacteriaceae,and Inc X3 plasmids may facilitated its spread.blaIMI-producing R.ornithinolytica was also first identified.We identified and completely sequenced the blaIMI-3 gene located on plasmids.blaIMI-3 was embedded in a novel transposon(Tn6306),which may be responsible for mobilization of the resistance gene.