NDM-5 carbapenemase was mainly identified in Escherichia coli, while the rapid transmission of bla_NDM-5 among Enterobacteriaceae has raised serious public attention. This study identified 14 NDM-5-producing Klebsiella pneumoniae isolates from 107 carbapenem-resistant K. pneumoniae isolates, recovered from blood, urine, and normally sterile body fluids of pediatric patients from January 2016 to December 2018. All NDM-5-producing isolates were highly resistant to β-lactams, while tigecycline and polymyxin B exhibited excellent antimicrobial activity. These 14 strains belonged to 9 different sequence types (STs) and displayed various pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they were not clonally related. S1-PFGE followed by Southern blotting showed that the bla_NDM-5 gene was located on an ∼46-kb IncX3 plasmid in all strains. All bla_NDM-5-carrying plasmids were successfully transferred into recipient E. coli J53. PCR-based sequencing demonstrated that all of the bla_NDM-5-carrying plasmids shared highly similar backbones, with nucleotide sequence identity of >99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 bla_NDM-5-carrying plasmids, with dynamic changes observed only in bla_NDM-5-surrounding elements. Interestingly, the IncX3 bla_NDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% bla_NDM-5-carrying plasmid loss after coculture with linoleic acid for 5 days. These results showed that the IncX3 plasmid facilitated the dissemination of bla_NDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniae.

中文翻译：

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blaNDM-5携带IncX3型质粒在肺炎克雷伯菌多克隆分离株中的传播及稳定性

NDM-5碳青霉烯酶主要在大肠杆菌中鉴定，而bla _NDM-5在肠杆菌中的快速传播引起了公众的严重关注。本研究中鉴定14 NDM -5-产生肺炎克雷伯菌从107碳青霉烯类耐药株肺炎克雷伯氏菌从 2016 年 1 月至 2018 年 12 月从儿科患者的血液、尿液和正常无菌体液中回收的分离株。所有产生 NDM-5 的分离株都对 β-内酰胺类具有高度耐药性，而替加环素和多粘菌素 B 表现出优异的抗菌活性。这 14 个菌株属于 9 个不同的序列类型 (ST)，并显示出各种脉冲场凝胶电泳 (PFGE) 模式，表明它们没有克隆相关性。S1-PFGE 和Southern 印迹表明bla _NDM-5基因位于所有菌株中的~46-kb IncX3 质粒上。所有bla _NDM-5携带质粒都成功转移到受体大肠杆菌J53 中。基于 PCR 的测序表明所有bla携带_{NDM-5 的}质粒共享高度相似的骨架，核苷酸序列同一性 >99%。此外，该质粒显示出与之前报道的流行病 IncX3 bla _NDM-5携带质粒的高度序列相似性，仅在bla _NDM-5周围元件中观察到动态变化。有趣的是，当在不含抗生素的培养基中培养时，携带 IncX3 bla _{NDM-5 的}质粒在临床分离株中表现出很强的稳定性。然而，在加入偶联抑制剂亚油酸后，可以观察到 IncX3 质粒丢失水平的逐渐增加。临床分离株显示 10% 至 15% bla _NDM-5- 与亚油酸共培养 5 天后携带质粒丢失。这些结果表明，IncX3质粒促进传播BLA _NDM-5多克隆中肺炎克雷伯菌儿童菌株和接合转移显著促成内IncX3质粒稳定性肺炎克雷伯。