耐碳青霉烯类大肠埃希菌对替加环素异质性耐药的机制

Mechanisms of Tigecycline Heteroresistance in Carbapenem⁃Resistant Escherichia coli

目的研究耐碳青霉烯类大肠埃希菌(CREC)对替加环素异质性耐药的分子机制,为临床治疗和合理应用抗生素提供依据。方法收集2017年1月至2019年3月间多中心分离的CREC,用改良E-test法和菌群谱型分析微量滴定法(MPAP)初筛,群体谱分析法(PAP)确认CREC对替加环素的异质性耐药情况,并用荧光定量RT-qPCR分析外排泵相关基因、外排泵调节基因和膜孔蛋白基因的表达。结果改良E-test法和MPAP法筛查CREC对替加环素异质性耐药(CREC-TGC-HR)的阳性率分别为25.0%(14/56)和39.3%(22/56)。经PAP法确认,有37.5%(21/56)为阳性。改良E-test法的CREC-TGC-HR检出率低于PAP法,差异有统计学意义(P<0.05)。3家医院CREC-TGC-HR的检出率分别是43.8%(7/16)、43.5%(10/23)和23.5%(4/17),两两比较差异均无统计学意义(P>0.05)。与CREC-TGC-HR原始菌株相比,耐药亚群外排泵基因acrA、acrB表达分别上调1.79倍、5.01倍;其相关调控基因marA、marB表达分别上调5.38和2.18倍;膜孔蛋白基因ompC表达下调2.35倍,差异均有统计学意义(P<0.05)。膜孔蛋白基因ompF和ompX的表达比较差异无统计学意义(P>0.05)。结论首次报道CREC对替加环素异质性耐药,且异质性耐药率较高,需要采用有效的方法检测CREC-TGC-HR,为临床治疗和合理应用抗生素提供依据。

Objective To study the molecular mechanism of Carbapenem⁃Resistant Escherichia coli(CREC)'s heteroresistance(HR)to tigecycline.Methods All strains were collected from January 2017 to March 2019.HR to tigecycline(TGC)was detected by modified E⁃test,Microtitration population analysis profiling(MPAP)and confirmed by population analysis profiling(PAP),and the expressions of efflux pump related genes and porin genes were analyzed by RT⁃qPCR.Results The positive rates of modified E⁃test and MPAP for CREC⁃TGC⁃HR were 25.0%(14/56)and 39.3%(21/56),respectively.Confirmed by PAP,A high prevalence 37.5%(21/56)of HR to tigecycline was observed in this study.The difference of positive rates between E⁃test and PAP was statistically significant(P<0.05).The detection rates of 3 Hospital were 43.8%(7/16),43.5%(10/23)and 23.5%(4/17),respectively.There was no statistically significant differ⁃ence(P>0.05).Mechanistic analysis revealed that efflux gene(acrA、acrB),the regulatory genes(marA,marB),porin gene(ompC,ompF)overexpression and ompX down⁃regulation contributed to the development of this phenomenon.Conclusion We firstly reported the phenotype of tigecycline HR,which has a high prev⁃alence in CREC.It is necessary to adopt an effective method to detect CREC⁃TGC⁃HR to provide a basis for clinical treatment and rational use of antibiotics.