The Resistance Mechanism And Molecular Epidemiology Of Carbapenem-resistant Klebsiella Pneumonia

Objective:Carbapenem-resistant Klebsiella pneumonia（CRKP）has become established as a major public concern.Our study aims at determining antimicrobial susceptibility,resistance mechanisms and spreading mechanisms of CRKP isolated from Shanghai Children’s Hospital in China,to provide advice for controlling the large-scale dissemination of CRKP.Methods:1.12,22 and 70 CRKP were collected from January to December in 2013,from March to June in 2014,from June 2014 to June 2015,respectively.General clinical data of patients were retrospectively reviewed.2.Antimicrobial susceptibility testing was determined by agar dilution method.Combined-disk assays using ertapenem with and without 3’-aminophenylboronic acid（APB）or EDTA were respectively used to phenotypically screen for class A or class B metallo-β-lactamase carbapenemases（MBL）.The presence of genes encoding carbapenemases KPC,IMI,NDM,IMP,VIM,and OXA,CTX-M type extended spectrumβ-lactamases（ESBLs）and genes encoding the plasmid-borne AmpCβ-lactamases were investigated.PCR amplicons were sequenced and the DNA sequences obtained were compared with those available in the NCBI GenBank database using BLAST searches.3.Clonal relatedness was identified by multilocus sequence typing（MLST）and pulsed field gel electrophoresis（PFGE）.Seven housekeeping genes（rpoB,gapA,mdh,Pgi,phoE,infB and tonB）were amplified by PCR and sequence type（ST）were obtained by sequencing seven housekeeping genes and submitting sequences to Klebsiella pneumonia MLST website.PFGE of XbaI-digested genomic DNA were performed as previously described and the results were analysed according to the criteria proposed by Tenover et al.Conjugation experiment was carried out to determine the transferability of carbapenemase gene.Whole-cell DNA of clinical strains embedded in agarose gel plugs,digested with S1 nuclease,was separated by PFGE.Plasmids obtained by PFGE were transferred to nylon membranes and hybridized with digoxigenin-labeled bla _NDM-1-specific probes.Results:Most of these strains were extensively drug resistant（XDR）bacteria.The resistance rate of cephalosporin and carbapenems is high.The resistance rate of imipenem of the three parts of strains were 91.7%,59.1%and 98.5%,while none were resistant to tigecycline and colistin.EDTA synergy test and APB synergy test showed good sensitity and specificity.The main mechanisms to carbapenems were producing NDM-1 or KPC carbapenemase.Among the three parts of strains,8.3%（1/12）,4.5%（1/22）and 15.7%（11/70）were identified as bla_KPC-2 positive while 75%（8/12）,77.3%（17/22）and 78.6%（55/70）were identified as bla_NDM-1 positive.ST11（58.3%,7/12）was predominant sequence type among the first part of strains.Apart from ST11,we have also detected ST278（n=2）,T37（n=1）,ST76（n=1）and ST610（n=1）isolates.ST37（50%,11/22）and ST76（36.4%,8/22）were predominant sequence types among the second part of strains.Apart from K.pneumonia ST37 and ST76,we have also detected ST846（n=1）,ST11（n=1）and ST571（n=1）.ST37（68.6%,48/70）was predominant sequence type among the third part of strains.Apart from ST37,we have also detected ST76（n=7）,ST11（n=9）,ST258（n=2）,ST571（n=1）,ST610（n=1）and ST846（n=2）.PFGE results showed that the first part of strains were grouped into 4 types and 3 sub-types（A₁,A₂,A₃,B,C,D）and ST11isolates were mainly PFGE-A₁.The second part of strains among were grouped into 5types（E,F,G,H,I）and ST76 and ST37 isolates were mainly PFGE-E and PFGE-G,respectively.The third part of strains among were grouped into 7 types（E,G,I,J,K,L,M）and ST37 isolates were mainly PFGE-E.There were clone-spread of several PTs among these strains.Conjugation experiments showed that plasmids bearing resistance genes in 86.4%（19/22）isolates was successfully transferred to E.coli J53or EC600.The transconjugants showed resistance to carbapenems.S1-PFGE revealed that the 15 bla _NDM-1-positive K.pneumonia harbored 1–4 plasmids.The hybridization results revealed that plasmids carrying bla_NDM-1 were approximately 50–240 kb in size.PBRT resuts showed 46.7%（7/15）bla_NDM-1-bearing plasmids belonged to Inc Frep.Conclusion:1.Carbapenem resistance mechanism of Klebsiella pneumonia turned from KPC-producing to NDM-1-producing from 2013 to 2015.NDM-1-producing K.pneumoniae have been widely disseminated in the hospital,so it is vitally important to monitor the resistant bacteria.2.While ST11 was dominant clone among KPC-producing isolates in 2013,ST76 and ST37 were main clones among NDM-1-producing strains isolated between 2014 and 2015.All these two STs（ST76and ST37）were different from STs reported recently home and abroad.The resistance genes were mainly located in Inc Frep plasmids different from previously reported which could disseminate between different strains,so it is necessary to intervene of strict infection-control measures,including proper hand hygiene,contact precautions and cohort nursing care,to reduce the cross infection and avoid the rapid spread or clonal dissemination of carbapenemase-producing Enterobacteriaceae strains in healthcare facilities.