Study On The Molecular Mechanism Of Carbapenem Heteroresistance In Pseudomonas Aeruginosa And Escherichia Coli

Objective:Heteroresistance is the intermediate stage and the necessary process for bacterial progression to full drug resistance,however,its mechanism remains unclear.This study aim to investigate the molecular epidemiology,the mechanism,and the risk factors for the acquisition of heteroresistance to cabapenems invasive P.aeruginosa infections and the mechanism of carbapenem heteroresistance in E.coli strains respectively,so as to better understand the occurrence and the development of carbapenem heteroresistance in different bacteria.Methods:A total of 451 non-duplicate P.aeruginosa strains recovered from the sterile body fluids were retrospectively collected from January 2011 to December 2015 in the First Affiliated Hospital of Chongqing Medical University.Drug susceptibility test was performed by the Vitek 2 Compact system,heteroresistance phenotype was preliminarily screened by Kirby-Bauer disk diffusion test(K-B test)and then verified by population analysis profile(PAP)and Time-Killing test.The gene homology of carbapenem-heteroresistant strains was detected by Pulsed Field Gel Electrophoresis(PFGE).MIC values for the native strains and their respective mutant subpopulations were detected by broth microdilution method.Expression levels of the efflux pump genes,blaAmpc and OprD genes for the native strains and their respective mutant subpopulations were detected by quantitative Real-time PCR(RT-qPCR).Meanwhile,all the clinical data were collected to identify the risk factors among invasive carbapenem-heteroresistant P.aeruginosa(CHPA)isolates by SPSS.21 and to analyze the correlation between carbapenem consumption and the incidence of CHPA isolates.In addition,Imipenem-heteroresistant phenotype was preliminarily screened by K-B test and then verified by PAP.Carbapenem-associated resistant genes were amplified by polymerase chain reaction(PCR)and sequencd in N8591 strain,including Carbapenemase genes,Extended Spectrum Beta-Lactamases genes(ESBLs)and outer membrane proteins genes.The OmpF sequence in ATCC25922 was replaced by OmpF mutant sequence in N8591 by the gene replacement techniques and then proving.Results:1.The study results of carbapenem-heteroresistance among P.aeruginosa were as follows:(1)During the investigation period,the drug susceptibilities of these 451 non-duplicate P.aeruginosa strains were re-evaluated by K-B,and the results of drug susceptibilities were respectively 245(54.3%)heteroresistant strains,126(27.9%)sensitive strains,69(15.3%)resistant strains and 11(2.5%)intermediate strains to IPM.Meanwhile,there were respectively 327(72.5%)heteroresistant strains,76(16.9%)sensitive strains,40(8.7%)resistant strains and 8(1.9%)intermediate strains to MEM.The higher percentage of heteroresistance was observed for MEM(72.5%),and followed by IPM(54.3%),and the prevalence of IPM-HR and MEM-HR were increasing yearly.(2)The native isolates and their mutant subgroups grown in the highest concentration of PAP had the same clone profile for single HR isolate by PFGE.Of the 99 clinical CHPA strains,they were clonal diversities,and there were also no three or more strains with the same clone profile of PFGE.(3)Among the possible mechanisms of CHPA isolates,MIC values for native isolate and their mutant subgroups were 8-fold or 16-fold difference.RT-qPCR showed that expression of the blaAMpc gene in IPM-HR and MEM-HR subpopulations increased from 1.04-1.32-fold and from 1.14-1.43-fold higher than that for native isolates,respectively,but the difference was not statistically significant(P>0.05).Moreover,Compared with their native isolates,expression levels of efflux pump genes in all the mutant subgroups were up-regulated,and the expression levels of mexB and mexE for IPM(increasing both from 1.58-1.78-fold and from 1.96-2.64-fold)and of mexB for MEM(increasing from 1.74-2.12-fold)was observed respectively to be significant(P<0.05).Furthermore,expression levels of the oprD gene in these strains was also detected to reduce from 0.56-0.12-fold(P<0.05).(4)Multivariate analysis showed that male sex,drainage tube and prior carbapenem therapy were independent risk factors associated with IPM-HR strains.Transfer from another hospital and prior carbapenem therapy were independent risk factor associated with MEM-HR strains.Moreover,prior carbapenem therapy was a common independent risk factor for the acquisition of IPM-HR and MEM-HR isolates.A positive correlation between heteroresistance rates to IPM and consumption of carbapenems in P.aeruginosa showed statistical significance(P<0.001,r2=0.941),and the heteroresistance rates to MEM had a similar trend towards significance(P<0.001,r2=0.918).2.Results of the resistant mechanism of carbapenem heteroresistance among E.coli were as follows:(1)The result of K-B test showed that the N8591 isolate was IPM-HR strian and then confirmed by PAP.The strain was resistant to most antibiotics,including amtrazine,ciprofloxacin,gentamicin,the third-generation and fourth-generation cephalosporins,however,it was the low level resistance to ETP(MIC values=2 ?g/mL)and sensitive to IPM.(2)The results of PCR showed that no carbapenemase genes were detected,the blacTx-M-3 gene were detected among ESBLs genes and a partial deletion(19 bp long)in the site 788 of OmpF gene had disturbed its reading frame and resulted the OmpF porin loss.(3)The OmpF sequence in ATCC25922 was successfully replaced by the OmpF mutant sequence in N8591.Imipenem-heteroresistance phenotype for ATCC25922/WompF isolate(replaced strain)was also found by K-B test and then verified by PAP.OmpF gene was amplified and sequenced in four randomly selected IPM-HR isolates,and the mutant sequence in the same location of OmpF gene in N8591 isolate was not found in above four IPM-HR strains.Conclusions:Heteroresistance is widespread in a variety of microbes,and there may be various molecule mechanisms of carbapenem-heteroresistance.1.Among investigation of the molecule mechanism of CHPA isolates,the rates of IPM-HR and MEM-HR were very high,and these CHPA isolates excluded the possibility of an outbreak by PFGE.Down-regulated expression of OprD gene and Up-regulated expression of efflux-associated genes should be the main molecule mechanism of CHPA strains.Moreover,prior carbapenem therapy was a common independent risk factor for the acquisition of CHPA isolates.2.OmpF porin loss may be one of the mechanisms of IPM-HR in E.coli.