Molecular Mechanism Of Carbapenem Heteroresistance Among Escherichia Coli And Evaluation Of Six Phenotypic Methods For The Identification Of Carbapenemases

Objective: To screen and verify the carbpenem-heteroresistant Escherichia coli(CHEC)isolates and explore its molecular mechanism and evaluate six phenotypic methods for the identification of carbapenemases.Methods: A total of 532 non-duplicate E.coli strains isolated from the sterile body fluids were collected from June 2011 to December 2015 in our hospital.CHEC isolates were screened by K-B test and verified by PAP.IPM MIC values were determined by broth microdilution method.Carbapenemase genes,ESBLs genes,OMP genes and mrd A were amplified by PCR and sequencing.The evolution of carbapenem heteroresistance was verified by IPM-induced test in vitro.Expression levels of blaTEM-1 and OMPs genes were detected by RT-qPCR.A total of256 gram-negative bacterial were detected by Modified Hodge test(MHT),Triton Hodge test(THT),Carba NP test(CNPt),simplified Carba NP test(CNPt-direct),blue-Carba NP test(BCT)and carbapenem inactivation method(CIM)for the identification of carbapenemases,and the sixphenotypic methods were evaluated by the McNemar’s test.Results: 1.CHEC strains screened by K-B tests isolated from 2011 to2015 were respectively 41(68.33%),41(34.17%),37(32.17%),70(59.32%)and 84(70.58%)heteroresistant to IPM,31(51.67%),29(24.17%),17(14.78%),30(25.42%)and 56(47.58%)heteroresistant to ETP,2(3.33%),8(6.67%),4(3.48%),12(10.17%)and 8(6.72%)heteroresistant to MEM.PAP test demonstrated all of the 36 phenotypic heteroresistant isolates were carbpenem-heteroresistant ones.2.Of the 36 CHEC isolates,33(91.67%)harbored blaCTX-M and12(33.3%)harbored blaTEM-1,10(27.8%)harbored a combination of blaCTX-M and blaTEM-1.Only one strain losed the OmpC gene.No carbapenemase genes were detected.3.In vitro IPM-induced test,under long-time and low concentration of IPM,subpopulations with higher MIC value emerged.PAP line chart exhibited a right shift toward higher carbapenem-resistance levels.It indicated carbapenem resistance evolved through heteroresistance as an intermediate stage.4.RT-qPCR showed that up-regulated expression of blaTEM-1 and down-regulated expression of OMP genes were significantly related with higher MIC values of subpopulations.5.A total of 256 gram-negative bacterial isolates,including 139 carbapenemase producers and 117 non-carbapenemase producers,weredetected by PCR and sequencing.Of the 139 carbapenemase producers,105(75.5%),118(84.9%),92(66.2%),129(92.8%),114(82.0%)and130(93.5 %)isolates respectively detected by MHT,THT,CNPt,CNPt-direct,BCT and CIM.CNPt-direct and CIM yielded comparable sensitivities(92.8% versus 93.5%,P=1),which were higher than that of MHT,CNPt and BCT(P<0.003).The specificity of THT(91.5%)was significantly lower than that(100%)of the four methods(CNPt,CNPt-direct,BCT and CIM),respectively(P <0.003).Conclusions: 1.Our study demonstrated that carbpenem heteroresistance is widely existed among clinical E.coli isolates.Although the heteroresistance rate of MEM was stable and low,the heteroresistance rates of IPM and ETP had an increased trend in recent three years.2.Carbapenem heteroresistance is the intermediate stage for evolution of carbapenem resistance in E.coli isolates.3.Up-regulated expression of blaTEM-1 and down-regulated expression of OMP genes might be the main mechanism for the evolution of carbapenem heteroresistance to carbapenem resistance in E.coli isolates.4.In our study,CNPt-direct and CIM possessed high sensitivity and specificity.They had great clinical application value for rapid and efficient detection of carbapenemase among the six phenotypic methods.