Characterization And Mechanism Of Tigecycline Heteroresistance In Carbapenem-resistant E.Cloacae

Background : Enterobacter cloacae is a normal intestinal flora of human.It is also an important conditional and nosocomial pathogen,causing clinical symptoms such as urinary tract infections,lower respiratory tract infections,bacteremia,and abdominal infections.Currently,with the global prevalence and outbreak of carbapenem-resistant Enterobacteriaceae,more advanced and ideal antibiotics are needed to treat infections of such bacteria.Tigecycline,as the first glycylcycline antibacterial agent,bind bacteria 30 s ribosome and inhibit protein synthesis.It has strong antibacterial activity against Gram-negative bacilli producing extended-spectrum ?-lactamases(ESBLs),AmpC and MBL enzymes,which is considered as one of the last resort for the treatment of carbapenem-resistant bacteria.However,recently the emergence of tigecycline-resistant strains has been reported globally,and the main mechanism of resistance is the over-expressed RND efflux pumps.Among them,the overexpression of AcrAB efflux pump was involved in tigecycline resistance in Escherichia coli,the overproduction of AcrAB combined with OqxAB efflux pump resulted in resistance of Klebsiella pneumonia and Enterobacter cloacae to tigecycline.In addition,mutations in the efflux pump regulatory genes RamA,MarA,and SoxS can also lead to overexpression of efflux pumps,further causing bacterial resistance totigecycline.Heteroresistance is an intermediate stage of bacteria from sensitive to fully resistant.Numerous reports have reported that bacteria exhibit heteroresistance to different kinds of antibiotics,which is of great significance for the research and evolution of bacterial resistance.However,heteroresistance is often not enough valued by clinicians and microbiologists,leading to failure and progression of antibiotic treatment.At present,there is no relevant study on the heteroresistance of carbapenem-resistant tigecycline in E.cloacae.Objective:1.To confirm the prevalence of tigecycline heteroresistance in clinical carbapenem-resistant E.cloacae strains.2.To explore the mechanism of tigecycline heteroresistance in E.cloacae strains.3.To detect the molecular epidemiology of tigecycline heteroresistance in E.cloacae.4.To assess the risk factors and clinical outcomes of tigecycline-heteroresistant E.cloacae infections.Method:1.Carbapenem-resistant E.cloacae strains were isolated in the First Affiliated Hospital of Chongqing Medical University from January 2014 to December 2017.The MIC values of E.cloacae on different antibiotics were screened and confirmed by vitek2 or micro-broth dilution method.2.K-B disk diffusion method,E-test test strip method,PAP test,Time-Killing test,growth experiment and passage stability test were used to detect and verify tigecycline heteroresistance phenotype.3.Efflux-mediated heteroresistance mechanisms were evaluated using the efflux pump inhibitor CCCP or PA?N,as well as expression of effluxpump genes(acrA,acrB,oqxA,and oqxB)and their regulators(soxS and ramA).4.PFGE and MLST typing were conducted to explore molecular epidemiological characteristics of tigecycline heteroresistance E.cloacae strains.5.Clinical data were collected and logistic regression was performed to analyze the independent risk factors and clinical prognosis of tigecycline-resistant E.cloacae infection.Result:1.From January 2014 to December 2017,a total of 140carbapenem-resistant and tigecycline sensitive E.cloacae strains were isolated.20%(28/140)strains were identified as TH-CRECL by using KB-test,E-test and PAP experiments.No significant difference in the growth rate between the clones inside and outside the K-B paper of TH-CRECL strain,and the tigecycline heteroresistance phenotypes were stably reproduced after passage growth.2.CCCP efflux pump inhibitors could not inhibit the resistance of TH-CRECL to tigecycline.PA?N efflux pump inhibitors could reverse the tigecycline MIC value of TH-CRECL strain.Fluorescence quantification displayed that the mechanism of TH-CRECL resistance was associated with overexpression of AcrAB and OqxAB efflux pumps and their transcriptional regulators(soxS and ramA).3.The original strains and their resistant subpopulations displayed similar PFGE band patterns,but all the 28 TH-CRECL strains had a total of21 different PFGE profiles.MLST genotyping results showed that ST97(4/28,14.3%),ST177(3/28,10.7%)and ST93(2/27,7.14%)dominated.Three new MLST types were found,ST1075,ST1076 and ST1077.4.Univariate analysis exhibited that transferring from other hospitals,admission to ICU,solid tumors,respiratory diseases,respiratory infections,trachea cannula and previous use of fluoroquinolone antibiotics were possible risk factors for infection with TH-CRECL.Logistic regression multivariate analysis showed that previous use of fluoroquinolone agent was the only potential independent risk factor for infection with TH-CRECL.Moreover,TH-CRECL infection may lead to a remarkably longer hospital stays and deterioration in functional status.Conclusion:We firstly discovered and reported the phenotype of tigecycline heteroresistance,which has a relatively low rate in carbapenem-resistant E.cloacae.Twenty-eight TH-CRECL isolates showed scattered distribution in molecular epidemiology and did not exhibit epidemic outbreak.The heteroresistance mechanism of these strains may be associated with overexpression of AcrAB and OqxAB efflux pumps.Previous treatment with fluoroquinolone antibiotic was identified as the only potential independent risk factor for infection with TH-CRECL.In addition,patients infected with the TH-CRECL strain may lead to prolonged hospital stay and deterioration in functional status.Therefore,timely detection and intervention of patients infected with TH-CRECL isolates has important clinical significance,and it is urgent to prevent and control the spread of such nightmare bacteria.