Study On The Mechanism Of Cabapenem Heteroresistance In Klebsiella Pneumoniae And Escherichia Coli

Objective:Heteroresistance?HR?refers to the phenomenon that different clonal subgroups have different antibiotic susceptibility under the same genetic background.As a special form of full drug resistance,heteroresistance is considered to be the intermediate stage for bacterial progression to the full drug resistance.This study analyzed the clinical characteristics,clinical outcomes and independent risk factors for the acquisitionofinfectionsbyCefepime-heteroresistant-Klebsiella pneumoniae?FEP-HR-KP?andErtapenem-heteroresistant-Klebsiella pneumoniae?ETP-HR-KP?,as well as the molecular mechanism of carbapenem heteroresistance in Escherichia coli?CHEC?,so as to provide relevant theoretical basis and control measures for clinical treatment.Methods:A retrospective case-control study was conducted on 108strains of K.pneumoniae isolated from the aseptic fluid samples of inpatients in the First Affiliated Hospital of Chongqing Medical University from January 2014 to December 2015.The Kirby-Bauer disk diffusion test?K-B test?and E-test were first used for the screening of heteroresistance phenotype,and then identified by the Population Analysis Profile?PAP?;The homology and molecular epidemiological characteristics analysis of heteroresistant strains were detected by Pulsed Field Gel Electrophoresis?PFGE?;Then we analyzed clinical characteristics of FEP-HR-KP and ETP-HR-KP within 2 years,and clinical data was used to analyze the independent risk factors associated with FEP-HR-KP and ETP-HR-KP infection through the SPSS.21 software.In addition,for the special case found in the study,K-B test and PAP were performed to screen and confirm the carbapenem-heteroresistance phenotype in E.coli,polymerase chain reaction?PCR?was conducted to detect carbapenem resistance-related genes,then we compared the virulence of three strains in vivo and vitro;The second-generation sequencing technology was carried out to achieve the whole-genome sequencing of the three strains,and clarified relevant information in the genetic level,then we completed the SNP mutation site verification of the efflux pump related genes.Results:1.The study results of epidemiological and risk factors of heteroresistance to cefepime and ertapenem among K.pneumoniae were as follows:?1?FEP-HR-KP and ETP-HR-KP were screened by K-B and E-test and then confirmed by PAP.?2?PFGE results showed that there was no difference between the bands of the primary and clonal subgroups of the heteroresistant strains,and there was a high degree of homology in the genetics,which could be considered as the same strain;However,the PFGE bands among the clinical strains were different,and there were 7 or more different bands between two isolates,which indicated there was no outbreak between the strains.?3?The distribution of drug susceptibility results of K.pneumoniae to FEP was as follows:51?51/108,47.2%?heteroresistant strains,29 sensitive strains?29/108,26.9%?,3 intermediate strains?3/108,2.8%?and 25 resistant strains?25/108,23.1%?to FEP;The distribution of drug susceptibility results of K.pneumoniae to ETP was as follows:63 heteroresistant strains?63/108,58.3%?,16 sensitive strains?16/108,14.8%?,4 intermediate strains?4/108,3.7%?and 25 resistant strains?25/108,23.2%?to ETP.Further analysis of the overall heteroresistant rate from 2014 to 2015 showed that the heteroresistant rate of strains to ETP was 58.3%?63/108?,which higher than that of FEP?51/108,47.2%?.Besides,the rate of heteroresistance has increased year by year.K.pneumoniae was mainly isolated from blood samples?36/108,33.3%?,and more than half of the specimens were from surgery?70/108,64.8%?.?4?Risk factors analysis showed soft tissue infection?OR:1.92,95%CI:0.91-3.91,P=0.049?,mechanical ventilation?OR:2.45,95%CI:1.31-4.47,P=0.013?and prior cephalosporins therapy?OR:2.10,95%CI:1.18-3.13,P=0.020?wereindependentriskfactorsfor FEP-HR-KP-associated infection?P<0.05?;Prior cephalosporins therapy?OR:2.08,95%CI:0.96-4.20,P=0.033?,drainage tube?OR:2.76,95%CI:1.49-4.90,P=0.018?,prior carbapenem therapy?OR:2.52,95%CI:1.08?-3.86,P=0.023)were independent risk factors for ETP-HR-KP-associated infection?P<0.05?.2,Results of the heteroresistant mechanism of carbapenem heteroresistance among E.coli were as follows:?1?We collected drug susceptibility information of the three strains,screened and confirmed heteroresistant phenotype through K-B test and PAP,respectively.We confirmed that 1001 was a susceptible strain,1021 was a heteroresistant strain and 88 was a drug-resistant strain.?2?The results of drug resistance-related genes showed that the strain No.1001 expressed bla_CTX-M-14TX-M-14 gene,the strain No.1021 expressed bla_CTX-M-14,bla_CMY-42MY-42 gene,and the strain No.88 expressed bla_CTX-M-15,bla_CMY-42,bla_OXA-1XA-1 gene,none of the them were detected the carbapenemase gene membrane pore protein.The virulence of the three strains in vitro and in vivo was consistent,which showed No.1021 strain had the strongest virulence,next was No.1001strain,and the No.88 strain had the weakest virulence.?3?SNP mutation site verification results showed there existed the amino acid substitution phenomenon among heteroresistant strains,which was D80L?80th from aspartic acid to leucine?and S81Q?81th from serine to glutamine?in the acrA gene,T50W?the 50th position from threonine to tryptophan?in the marA gene,and L97E?the 97th position from leucine to glutamic acid?in the acrR gene,V57H?57th from proline to histidine?in the soxR gene.Conclusion:Heteroresistance is a special form of drug resistance,which is common in the resistance of various bacteria to various types of antibiotics.The study results of epidemiological and risk factors of heteroresistance to cefepime and ertapenem among K.pneumoniae indicated that invasive procedures and long-term antibiotic treatment history would increase the risk of this heteroresistant strains associated infections in patients,therefore,reduce unnecessary invasive procedures and short-term combination therapy with multiple antibiotics would be effective measures to prevent and control the emergence of such strains.In addition,there are few studies about heteroresistance at China and abroad,the mechanism is still unclear.The related mechanisms of heteroresistant mechanism of carbapenem heteroresistance among E.coli were as follows:1.The expression of the gene correlated with ESBLs may be related to the heteroresistance to carbapenem among E.coli.2.Gene mutations related to efflux pumps may be the mechanism by which CHEC strains eventually evolved from sensitive to heteroresistance finally to drug resistance.