Transmission of carbapenem-resistant Enterobacterales (CRE) in hospitals has been shown to occur through complex, multifarious networks driven by both clonal spread and horizontal transfer mediated by plasmids and other mobile genetic elements. We performed nanopore long-read sequencing on CRE isolates from a large urban hospital system to determine the overall contribution of plasmids to CRE transmission and identify specific plasmids implicated in the spread of bla_KPC (the Klebsiella pneumoniae carbapenemase [KPC] gene). Six hundred and five CRE isolates collected between 2009 and 2018 first underwent Illumina sequencing for genome-wide genotyping; 435 bla_KPC-positive isolates were then successfully nanopore sequenced to generate hybrid assemblies including circularized bla_KPC-harboring plasmids. Phylogenetic analysis and Mash clustering were used to define putative clonal and plasmid transmission clusters, respectively. Overall, CRE isolates belonged to 96 multilocus sequence types (STs) encoding bla_KPC on 447 plasmids which formed 54 plasmid clusters. We found evidence for clonal transmission in 66% of CRE isolates, over half of which belonged to four clades comprising K. pneumoniae ST258. Plasmid-mediated acquisition of bla_KPC occurred in 23%–27% of isolates. While most plasmid clusters were small, several plasmids were identified in multiple different species and STs, including a highly promiscuous IncN plasmid and an IncF plasmid putatively spreading bla_KPC from ST258 to other clones. Overall, this points to both the continued dominance of K. pneumoniae ST258 and the dissemination of bla_KPC across clones and species by diverse plasmid backbones. These findings support integrating long-read sequencing into genomic surveillance approaches to detect the hitherto silent spread of carbapenem resistance driven by mobile plasmids.

中文翻译：

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纽约市一家医院对碳青霉烯类耐药肠杆菌类菌 10 年的基因组流行病学揭示了复杂的质粒克隆动力学和 blaKPC 频繁水平转移的证据


碳青霉烯类耐药肠杆菌 （CRE） 在医院中的传播已被证明是通过由质粒和其他可移动遗传元件介导的克隆传播和水平转移驱动的复杂、多样化的网络发生的。我们对来自大型城市医院系统的 CRE 分离株进行了纳米孔长读长测序，以确定质粒对 CRE 传播的总体贡献，并鉴定与 bla_KPC 传播有关的特定质粒 （肺炎克雷伯菌碳青霉烯酶 [KPC] 基因）。2009 年至 2018 年间收集的 605 株 CRE 分离株首先进行了 Illumina 测序以进行全基因组基因分型;然后成功地对 435 个 bla_KPC 阳性分离株进行纳米孔测序，以生成杂交组装体，包括循环化的 bla_KPC 携带质粒。系统发育分析和 Mash 聚类分别用于定义推定的克隆和质粒传播簇。总体而言，CRE 分离株属于 96 种多位点序列类型 （ST），在 447 个质粒上编码 bla_KPC，形成 54 个质粒簇。我们在 66% 的 CRE 分离株中发现了克隆传播的证据，其中一半以上属于包含肺炎克雷伯菌 ST258 的四个分支。质粒介导的 bla_KPC 获得发生在 23%-27% 的分离株中。虽然大多数质粒簇很小，但在多个不同物种和 ST 中鉴定出几种质粒，包括高度混杂的 IncN 质粒和推定将 bla_KPC 从 ST258 传播到其他克隆的 IncF 质粒。总体而言，这表明 K 继续占据主导地位。 pneumoniae ST258 和 bla_KPC 通过不同质粒骨架在克隆和物种中的传播。这些发现支持将长读长测序整合到基因组监测方法中，以检测由移动质粒驱动的碳青霉烯类耐药性迄今为止的无声传播。