肺炎克雷伯菌能够获得各种外源遗传元件并随后赋予高的抗微生物耐药性。最近,质粒介导的RND家族多药外排泵基因簇,tmexCD1-toprJ1在肺炎克雷伯菌中发现。在这项研究中,我们分析了2017年至2021年监测患者中对替加环素耐药的肺炎克雷伯菌分离株.除了表型检测,包括生长曲线,质粒的可转移性和稳定性,高粘膜粘度,生物膜的形成,和血清存活率,通过全基因组测序,我们分析了携带tmexCD1-toprJ1的分离株的系统发育关系,并发现了携带tmexCD1-toprJ1的质粒的组成。总的来说,我们发现,来自5例患者的12株对替加环素耐药的分离株具有tmexCD1-toprJ1,命名为序列类型22(ST22)和ST3691.ST11分离物带有部分tmexD1,而完整的toprJ1(tmexC1丢失)对替加环素敏感。所有抗ST22替加环素的分离株都包含tmexCD1-toprJ1,blaNDM-1和blaKPC-2。tmexCD1-toprJ1由ST22中的新型IncU质粒和ST3691中的IncFIB/HI1B质粒编码,它们在迁移率和稳定性方面存在差异。有趣的是,来自同一患者的分离株对替加环素表现出异质耐药,不仅在来自不同样本的分离物中,而且在来自同一样本的分离物中,这可能归因于tmexCD1-toprJ1的差异表达,这是由于将tmexCD1-toprJ1重新定位在质粒复制起点附近而引起的动态遗传异质性。这里,我们报道了同时携带tmexCD1-toprJ1,blaNDM-1和blaKPC-2的肺炎克雷伯菌分离株的出现.结果突出了tmexCD1-toprJ1携带元件的体内遗传异质性对替加环素抗性体内变异的影响,这可能对抗菌治疗有显著影响。IMPORTANCEPandrug耐药(PDR)肺炎克雷伯菌对公共卫生构成了巨大挑战,替加环素是抗菌治疗的重要选择。在这项研究中,我们报道了包含tmexCD1-toprJ1,blaNDM-1和blaKPC-2的PDR肺炎克雷伯菌的出现,它们属于ST22和ST3691.通过全基因组分析,我们通过获取编码tmexCD1-toprJ1或blaNDM-1的多个遗传元件,重建了ST22祖先的进化图,成为PDR超级细菌。重要的是,ST22分离株中tmexCD1-toprJ1的遗传背景是不同的,并且具有不同的迁移率和稳定性.此外,我们还发现了在ST22长期感染期间替加环素耐药性的异质性,这可能归因于tmexCD1-toprJ1的差异表达,这是由于tmexCD1-toprJ1在质粒复制起点附近重新定位引起的动态遗传异质性.这项研究追踪了超级细菌PDR肺炎克雷伯菌的宿主间和宿主内微观进化,并强调了在抗菌治疗期间及时监测病原体变化的重要性。
Klebsiella pneumoniae is capable of acquiring various exogenous genetic elements and subsequently conferring high antimicrobial resistance. Recently, a plasmid-mediated RND family multidrug efflux pump gene cluster, tmexCD1-toprJ1, was discovered in K. pneumoniae. In this study, we analyzed tigecycline-resistant K. pneumoniae isolates from patients from surveillance from 2017 to 2021. In addition to phenotype detection, including growth curves, plasmid transferability and stability, hypermucoviscosity, biofilm formation, and serum survival, by whole-genome sequencing, we analyzed the phylogenetic relationships of the isolates harboring tmexCD1-toprJ1 and discovered the composition of plasmids carrying tmexCD1-toprJ1. In total, we discovered that 12 tigecycline-resistant isolates from 5 patients possessed tmexCD1-toprJ1, designated sequence type 22 (ST22) and ST3691. An ST11 isolate harbored a partial tmexD1, and a complete toprJ1 (tmexC1 was lost) was tigecycline sensitive. All the ST22 tigecycline-resistant isolates coharbored tmexCD1-toprJ1, blaNDM-1, and blaKPC-2. tmexCD1-toprJ1 was encoded by a novel IncU plasmid in ST22 and an IncFIB/HI1B plasmid in ST3691, which presented differences in mobility and stability. Interestingly, isolates from the same patients presented heteroresistance to tigecycline, not only among isolates from different specimens but also those from the same sample, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. Here, we reported the emergence of K. pneumoniae isolates coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2. The results highlight the impact of in vivo genetic heterogeneity of tmexCD1-toprJ1-carrying elements on the in vivo variation of tigecycline resistance, which might have notable influences on antimicrobial treatment. IMPORTANCE Pandrug-resistant (PDR) Klebsiella pneumoniae poses a great challenge to public health, and tigecycline is an essential choice for antimicrobial treatment. In this study, we reported the emergence of PDR K. pneumoniae coharboring tmexCD1-toprJ1, blaNDM-1, and blaKPC-2, which belongs to ST22 and ST3691. By whole-genome analysis, we reconstructed the evolutionary map of the ST22 ancestor to become the PDR superbug by acquiring multiple genetic elements encoding tmexCD1-toprJ1 or blaNDM-1. Importantly, the genetic contexts of tmexCD1-toprJ1 among the ST22 isolates are different and present with various mobilities and stabilities. Furthermore, we also discovered the heterogeneity of tigecycline resistance during long-term infection of ST22, which might be attributed to the differential expression of tmexCD1-toprJ1 due to the dynamic genetic heterogeneity caused by relocating tmexCD1-toprJ1 close to the replication origin of plasmid. This study tracks the inter- and intrahost microevolution of the superbug PDR K. pneumoniae and highlights the importance of timely monitoring of the variation of pathogens during antimicrobial treatment.