Plasmids remain important microbial components mediating the horizontal gene transfer (HGT) and dissemination of antimicrobial resistance. To systematically explore the relationship between mobile genetic elements (MGEs) and antimicrobial resistance genes (ARGs), a novel strategy using single-molecule real-time (SMRT) sequencing was developed. This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant (MDR) Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period. The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates. The plasmid pool was then subjected to SMRT sequencing, and 82 assembled plasmid fragments were obtained. In total, 124 ARGs (responsible for resistance to β-lactam, fluoroquinolone, and aminoglycoside, among others) and 317 MGEs [including transposons (Tns), insertion sequences (ISs), and integrons] were derived from these fragments. Most of these ARGs were linked to MGEs, allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements. Key elements involved in resistance transposition were identified, including IS26, Tn3, IS903B, ISEcp1, and ISKpn19. As the most predominant IS in the network, a typical IS26-mediated multicopy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications (TSDs). The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs, underpinning the clinical value of dominant HGT profiles.

质粒仍然是介导水平基因转移 (HGT) 和抗菌素耐药性传播的重要微生物成分。为了系统地探索移动遗传元件 (MGE) 和抗菌素耐药基因 (ARG) 之间的关系，开发了一种使用单分子实时 (SMRT) 测序的新策略。这种方法适用于来自临床分离的耐多药 (MDR)肺炎克雷伯菌的混合接合质粒从三级转诊医院超过 9 个月。接合质粒库是从与 53 个临床分离株进行质粒接合后获得抗菌素耐药性的转接合体中获得的。然后对质粒库进行 SMRT 测序，获得 82 个组装的质粒片段。总共有 124 个 ARG（负责对 β-内酰胺、氟喹诺酮和氨基糖苷等的抗性）和 317 个 MGE [包括转座子 (Tns)、插入序列 (IS) 和整合子]来自这些片段。这些 ARG 中的大多数都与 MGE 相关联，从而允许在 MGE 和/或 ARG 之间建立关系网络，可用于描述移动元素对抵抗力的传播。确定了涉及抗性转座的关键要素，包括 IS 26、Tn 3、IS 903B、IS Ecp1和 IS Kpn19。作为网络中最主要的 IS，典型的 IS 26介导的多拷贝复合转座事件通过追踪其侧翼的 8 bp 目标位点重复 (TSD) 来说明。混合质粒序列的景观突出了 MGE 和 ARG 之间关系的多样性和复杂性，支撑了显性 HGT 谱的临床价值。