碳青霉烯类耐药肠杆菌目细菌对头孢他啶/阿维巴坦的耐药率及耐药机制研究

Study on Resistance Rate and Mechanisms of Ceftazidime/Avibactam Resistance in Carbapenem-Resistant Enterobacterales

目的:了解本院临床分离的碳青霉烯类耐药肠杆菌目细菌(CRE)对头孢他啶/阿维巴坦(CAZ/AVI)的药敏情况,并初步探讨其耐药机制。方法:收集2020年1月~2021年6月本院临床分离的CRE菌株,采用微量肉汤稀释法和纸片扩散法检测受试菌株对CAZ/AVI的敏感性。采用碳青酶烯酶抑制剂增强试验对其中32株细菌进行碳青霉烯酶表型检测,采用酶免疫层析法、PCR及测序方法对5种常见碳青霉烯酶基因型(bla_(KPC)、bla_(NDM)、bla_(IMP)、bla_(VIM)和bla_(OXA-48-like))进行检测,并采用实时荧光定量PCR方法对bla_(KPC-2)基因的相对表达量进行检测。结果:共检出1692株CRE菌株,对CAZ/AVI耐药的菌株共101株,整体耐药率为6.0%。其中,碳青霉烯耐药肺炎克雷伯菌(CR-KP)的耐药率仅为2.9%,大肠埃希菌的耐药率为30.1%。CAZ/AVI耐药菌株中,CR-KP(29株,28.7%)居首位,大肠埃希菌(25株,24.8%)次之。对32株CRE菌株进行表型试验发现,单产金属酶19株(59.4%),丝氨酸酶联合金属酶7株(21.9%),单产丝氨酸酶5株(15.6%),酶型阴性1株(3.1%)。进行酶免疫层析法、PCR及测序发现,产碳青霉烯酶最主要的基因是bla_(NDM-1)(12株,37.5%),其次是bla_(NDM-5)(7株,21.9%)、bla_(KPC-2)(5株,15.6%)、bla_(KPC-2)联合bla_(NDM-1)(5株,15.6%)、bla_(KPC-2)联合bla_(NDM-5)(2株,6.3%)以及bla_(KPC-33)(1株,3.1%)。与CAZ/AVI敏感的CR-KP菌株相比,5株CAZ/AVI耐药且单产KPC-2酶的CR-KP菌株的bla_(KPC-2)基因相对表达量增加4.24倍(P=0.0368)。结论:本院临床分离的CRE菌株对CAZ/AVI的耐药率整体较低,耐药菌株以肺炎克雷伯菌和大肠埃希菌为主。单产或联合产NDM型金属酶是CRE菌株对CAZ/AVI耐药的最主要原因,少数单产KPC酶的CR-KP菌株可能由于bla_(KPC-2)基因高表达或bla_(KPC-2)基因突变形成不同的KPC酶亚型而对CAZ/AVI耐药。

Objective:To analyze the susceptibility of ceftazidime/avibactam(CAZ/AVI)resistance in carbapenem-resistant Enterobacterales(CRE)isolated from our hospital and to preliminarily discuss the mechanism of drug resistance.Methods:CRE isolates from our hospital were collected from January 2020 to June 2021,and the sensitivity of the tested strains to CAZ/AVI was detected by broth microdilution method and K-B method.Carbapenem resistance phenotypes were detected by carbapenemase inhibitor enhancement test for 32 CRE strains.Colloidal gold immunochromatography assay,polymerase chain reaction(PCR)and gene sequencing were performed to detect five common carbapenem-resistant genes(bla_(KPC),bla_(NDM),bla_(IMP),bla_(VIM)and bla_(OXA-48-like)).The relative expression of bla_(KPC-2)gene was detected by real-time quantitative PCR(RT-q PCR).Results:A total of 1692 CRE isolates in our hospital were collected,including 101 isolates resistant to CAZ/AVI(6.0%).Among them,the drug resistance rate of carbapenem-resistant Klebsiella pneumoniae(CR-KP)was only 2.9%,and the drug resistance rate of Escherichia coli was 30.1%.Among CAZ/AVI resistant strains,CR-KP(29 cases,28.7%)ranked first,followed by Escherichia coli(25cases,24.8%).The phenotypic test of 32CRE strains showed that 19 isolates(59.4%)were producing metallo-β-lactamase(MBL),7 isolates(21.9%)coproducing MBL and serine-β-lactamase(SBL),5 isolates(15.6%)producing SBL and 1 isolate(3.1%)with a negative result.Colloid gold,PCR and sequencing showed that the most important gene of carbapenemase was bla_(NDM-1)(12 cases,37.5%),followed by bla_(NDM-5)(7 cases,21.9%),bla_(KPC-2)(5 cases,15.6%),bla_(KPC-2)combined with bla_(NDM-1)(5 cases,15.6%),bla_(KPC-2)combined with bla_(NDM-5)(2 cases,6.3%)and bla_(KPC-33)(1 cases,3.1%).The relative expression of bla_(KPC-2)in 5 CR-KP isolates resistant to CAZ/AVI and producing KPC-2 enzyme increased to 4.24 fold compared with that in CR-KP isolates susceptible to CAZ/AVI(P=0.0368).Conclusion:The drug resistance rate of CRE strains isolated in our hospital to CAZ/AVI was generally low,and the resistant strains were mainly Klebsiella pneumoniae and Escherichia coli.Single or combined production of metallo-β-lactamase(NDM)was the main cause of CAZ/AVI resistance.Overexpression of bla_(KPC-2)or mutations of blak PC-2 gene into KPC subtypes also lead to CAZ/AVI resistance in some CR-KP strains single producing in some CR-KP strains single producing KPC enzyme.