耐碳青霉烯类肺炎克雷伯菌对头孢他啶/阿维巴坦耐药机制的研究

Mechanisms of resistance to ceftazidime/avibactam of carbapenem-resis-tant Klebsiella pneumoniae

摘要:

目的探讨耐碳青霉烯类肺炎克雷伯菌(CRKP)的分子流行病学特点,揭示其对头孢他啶/阿维巴坦(CZA)的耐药机制。方法收集2021年1月—2023年9月徐州医科大学附属医院临床首次分离的CZA耐药CRKP,采用基因扩增法和胶体金法检测bla_KPC、bla_NDM、bla_OXA、bla_VIM和bla_IMP五种碳青霉烯酶基因携带情况,采用实时荧光定量聚合酶链反应(RT-qPCR)方法检测产肺炎克雷伯菌碳青霉烯酶肺炎克雷伯菌(KPC-KP)相对拷贝数以及表达量,采用全基因测序方法分析KPC突变株的突变位点,以分析CRKP流行特征及对CZA的耐药机制。结果共分离73株对CZA耐药的CRKP,其中37株(50.68%)为KPC+NDM联产菌株,33株(45.21%)为单产NDM的菌株(单产NDM-5 23株,单产NDM-1 10株),3株单产KPC的菌株。发现菌株KP-2842为ST11型KPC-33突变株;菌株KP-2127和KP-2189为产KPC-2菌株,与肺炎克雷伯菌ATCC BAA-1705相比,其bla_KPC拷贝量分别上调1.04~3.86倍,而表达量分别增加6.66~12.93倍;胶体金法与PCR结合双向测序方法两者结果一致性良好,同时可以覆盖联产酶及KPC-33突变体的检测。结论该院CRKP对CZA耐药机制主要由金属酶NDM介导,其中NDM、KPC联产是本地区CRKP的主要特点,部分菌株对CZA耐药可能由bla_KPC-2高拷贝和高表达导致,并在江苏地区的ST11型CRKP中首次发现了KPC-33突变体。

Abstract:

Objective To explore the molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP), and reveal its mechanism of resistance to ceftazidime/avibactam (CZA). Methods CZA-resistant CRKP strains initially isolated from the Affiliated Hospital of Xuzhou Medical University from January 2021 to September 2023 were collected. The carriage of 5 carbapenemase genes (bla_KPC, bla_NDM, bla_OXA, bla_VIM, bla_IMP) were detected with gene amplification method and colloidal gold method. The relative copy number and expression level of Klebsiella pneumoniae (KP) carbapenemase-producing KP (KPC-KP) was detected with real-time quantitative polymerase chain reaction (RT-qPCR), mutation sites of KPC mutation strains were analyzed with whole-genome sequencing, and epidemic characteristics of CRKP and resistance mechanism to CZA were analyzed. Results A total of 73 CZA-resistant CRKP strains were isolated, with 37 (50.68%) being KPC and NDM co-producing strains, 33 (45.21%) NDM-producing alone (23 strains producing NDM-5 and 10 strains producing NDM-1), and 3 KPC-producing alone. KP-2842 strain was identified as ST11-type KPC-33 variant, KP-2127 and KP-2189 strains produced KPC-2. Compared with KP ATCC BAA-1705, the copy number of bla_KPC in these strains up-regulated by 1.04-3.86 fold, and the expression increased by 6.66-12.93 fold, respectively. Colloidal gold and PCR methods demonstrated good consistency and the ability to detect the enzyme co-producing and KPC-33 variant. Conclusion In this hospital, the resistance of CRKP to CZA is primarily mediated by the metalloenzyme NDM, with co-production of NDM and KPC being a characteristic of CRKP. High copy number and expression level of bla_KPC-2 also contribute to CZA resistance. This study identified the KPC-33 variant for the first time in ST11-type CRKP in Jiangsu Province.