Department of Laboratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.
Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, China.
Sci Rep. 2024 Nov 5;14(1):26723. doi: 10.1038/s41598-024-78205-9.
Plasmids, as important genetic elements apart from chromosomes, often carry multiple resistance genes and various mobile genetic elements, enabling them to acquire more exogenous genes and confer additional resistance phenotypes to bacteria. Various carbapenem resistance genes are often located on IncN plasmids, and several reports have linked fusion plasmids to IncN plasmids. Therefore, this study aims to explore the emergence, molecular structure characteristics, and resistance features mediated by IncN fusion plasmids carrying multiple carbapenem resistance genes. In this study, species identification was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Polymerase chain reaction (PCR) was employed to detect the presence of carbapenem resistance genes in the strains. PCR-based replicon typing (PBRT) was used to identify IncN plasmids. Plasmids were analyzed through S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, conjugation experiments, and stability tests. Whole-genome sequencing (WGS) and antimicrobial susceptibility testing (AST) were conducted to characterize the target strains. Four strains containing IncN plasmids were identified: two Klebsiella pneumoniae, one Escherichia coli, and one Enterobacter cloacae, all harboring carbapenem resistance genes. Among them, two IncN plasmids (pFAHZZU7605-KPC-IMP and pFAHZZU7865-IMP) contained blaIMP-4 and exhibited similar molecular structure characteristics. Notably, the pFAHZZU7605-KPC-IMP plasmid harbored both IncN and IncR replicons. We hypothesize that the pFAHZZU7605-KPC-IMP fusion plasmid resulted from the recombination of a pFAHZZU7865-IMP-like plasmid and an IncR-like plasmid. Further analysis of the plasmid's genetic elements revealed that insertion sequences ISKpn19 and ISKpn27 played crucial roles in the plasmid recombination and fusion process. In clinical settings, plasmids carrying different resistance genes can undergo fusion, mediated by genetic elements, thereby expanding the resistance spectrum of host bacteria. Hence, it is essential to enhance the monitoring and research of transposable elements to control the spread of multidrug-resistant bacteria.
质粒是染色体外的重要遗传元件，常携带多种耐药基因和各种移动遗传元件，使它们能够获得更多的外源基因，并赋予细菌额外的耐药表型。各种碳青霉烯类耐药基因常位于 IncN 质粒上，已有报道称融合质粒与 IncN 质粒有关。因此，本研究旨在探讨携带多种碳青霉烯类耐药基因的 IncN 融合质粒的出现、分子结构特征和耐药特征。在本研究中，采用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF/MS）进行物种鉴定。采用聚合酶链反应（PCR）检测菌株中碳青霉烯类耐药基因的存在。采用基于 PCR 的复制子分型（PBRT）鉴定 IncN 质粒。通过 S1-核酸酶脉冲场凝胶电泳（S1-PFGE）、Southern 印迹、接合实验和稳定性测试分析质粒。进行全基因组测序（WGS）和抗菌药物敏感性试验（AST）以对靶菌株进行特征描述。鉴定出 4 株携带 IncN 质粒的菌株：2 株肺炎克雷伯菌、1 株大肠埃希菌和 1 株阴沟肠杆菌，均携带碳青霉烯类耐药基因。其中 2 株 IncN 质粒（pFAHZZU7605-KPC-IMP 和 pFAHZZU7865-IMP）携带 blaIMP-4，具有相似的分子结构特征。值得注意的是，pFAHZZU7605-KPC-IMP 质粒同时携带 IncN 和 IncR 复制子。我们假设 pFAHZZU7605-KPC-IMP 融合质粒是由 pFAHZZU7865-IMP 样质粒和 IncR 样质粒重组产生的。进一步分析质粒的遗传元件表明，插入序列 ISKpn19 和 ISKpn27 在质粒的重组和融合过程中发挥了关键作用。在临床环境中，携带不同耐药基因的质粒可通过遗传元件发生融合，从而扩大宿主菌的耐药谱。因此，加强对可移动遗传元件的监测和研究，对于控制多药耐药菌的传播至关重要。
