Infections with multidrug-resistant bacteria often leave limited or no treatment options. The transfer of antimicrobial resistance genes (ARG) carrying plasmids between bacterial species by horizontal gene transfer represents an important mode of expansion of ARGs. Here, we demonstrate the application of Nanopore sequencing in a hospital setting for monitoring transfer and rapid evolution of antibiotic resistance plasmids within and across multiple species. In 2009, we experienced an outbreak with extensively multidrug-resistant Pseudomonas aeruginosa harboring the carbapenemase-encoding bla_IMP-8 gene. In 2012, the first Citrobacter freundii and Citrobacter cronae strains harboring the same gene were detected. Using Nanopore and Illumina sequencing, we conducted comparative analysis of all bla_IMP-8 bacteria isolated in our hospital over a 6-year period (n = 54). We developed the computational platform plasmIDent for Nanopore-based characterization of clinical isolates and monitoring of ARG transfer, comprising de novo assembly of genomes and plasmids, plasmid circularization, ARG annotation, comparative genome analysis of multiple isolates, and visualization of results. Using plasmIDent, we identified a 40-kb plasmid carrying bla_IMP-8 in P. aeruginosa and C. freundii, verifying the plasmid transfer. Within C. freundii, the plasmid underwent further evolution and plasmid fusion, resulting in a 164-kb megaplasmid, which was transferred to C. cronae. Multiple rearrangements of the multidrug resistance gene cassette were detected in P. aeruginosa, including deletions and translocations of complete ARGs. In summary, plasmid transfer, plasmid fusion, and rearrangement of the ARG cassette mediated the rapid evolution of opportunistic pathogens in our hospital. We demonstrated the feasibility of near-real-time monitoring of plasmid evolution and ARG transfer in clinical settings, enabling successful countermeasures to contain plasmid-mediated outbreaks.

中文翻译：

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通过纳米孔测序追踪医院环境中的抗生素耐药性转移和快速质粒进化。

多重耐药细菌感染通常导致治疗选择有限或没有治疗选择。通过水平基因转移在细菌物种之间转移携带质粒的抗菌素耐药基因（ARG）是ARG扩增的重要模式。在这里，我们展示了纳米孔测序在医院环境中的应用，用于监测多个物种内部和跨物种的抗生素抗性质粒的转移和快速进化。2009 年，我们经历了一场带有碳青霉烯酶编码bla _IMP-8基因的广泛多重耐药铜绿假单胞菌的爆发。2012年，首次检测到含有相同基因的弗氏柠檬酸杆菌和克罗纳柠檬酸杆菌菌株。使用 Nanopore 和 Illumina 测序，我们对我们医院 6 年来分离的所有bla _{IMP-8细菌进行了比较分析 (} n = 54)。我们开发了计算平台plasmIDent，用于基于纳米孔的临床分离株表征和 ARG 转移监测，包括基因组和质粒的从头组装、质粒环化、ARG 注释、多个分离株的比较基因组分析以及结果可视化。使用plasmIDent ，我们在铜绿假单胞菌和弗氏梭菌中鉴定了携带bla _IMP-8的 40 kb 质粒，验证了质粒转移。在弗氏梭菌中，该质粒经历了进一步的进化和质粒融合，产生了 164 kb 的大质粒，并将其转移到克氏梭菌中。在铜绿假单胞菌中检测到多药耐药基因盒的多重重排，包括完整 ARG 的缺失和易位。综上所述，质粒转移、质粒融合和ARG盒重排介导了我院机会性病原体的快速进化。我们证明了在临床环境中近实时监测质粒进化和 ARG 转移的可行性，从而能够成功采取对策来遏制质粒介导的疫情爆发。