Combining sequencing approaches to fully resolve a carbapenemase-encoding megaplasmid in a <i>Pseudomonas shirazica</i> clinical strain

Horizontal transfer of plasmids plays a pivotal role in dissemination of antibiotic resistance genes and emergence of multidrug-resistant bacteria. Plasmid sequencing is thus paramount for accurate epidemiological tracking in hospitals and routine surveillance. Combining Nanopore and Illumina sequencing allowed full assembly of a carbapenemase-encoding megaplasmid carried by multidrug-resistant clinical isolate FFUP_PS_41. Average nucleotide identity analyses revealed that FFUP_PS_41 belongs to the recently proposed new species Pseudomonas shirazica, related to the P. putida phylogenetic group. FFUP_PS_41 harbours a 498,516-bp megaplasmid (pJBCL41) with limited similarity to publicly-available plasmids. pJBCL41 contains genes predicted to encode replication, conjugation, partitioning and maintenance functions and heavy metal resistance. The |aacA7|blaVIM-2|aacA4| cassette array (resistance to carbapenems and aminoglycosides) is located within a class 1 integron that is a defective Tn402 derivative. This transposon lies within a 50,273-bp region bound by Tn3-family 38-bp inverted repeats and flanked by 5-bp direct repeats (DR) that composes additional transposon fragments, five insertion sequences and a Tn3-Derived Inverted-Repeat Miniature Element. The hybrid Nanopore/Illumina approach allowed full resolution of a carbapenemase-encoding megaplasmid from P. shirazica. Identification of novel megaplasmids sheds new light on the evolutionary effects of gene transfer and the selective forces driving antibiotic resistance.质粒水平转移在抗生素抗性基因的传播以及多重耐药细菌的出现过程中发挥关键作用。因此，质粒测序对于医院内的精准流行病学追踪与常规监测而言至关重要。结合纳米孔（Nanopore）测序与Illumina测序技术，研究人员成功完成了一株携带碳青霉烯酶编码巨型质粒的多重耐药临床分离株FFUP_PS_41的完整组装。平均核苷酸同源性（Average nucleotide identity, ANI）分析结果显示，FFUP_PS_41隶属于近期提出的新种设拉子假单胞菌（Pseudomonas shirazica），该菌种与恶臭假单胞菌（P. putida）系统发育群具有亲缘关系。FFUP_PS_41携带一条长度为498516 bp的巨型质粒pJBCL41，该质粒与公开数据库中的现有质粒仅存在有限同源性。pJBCL41携带有预测可编码复制、接合、分配与维持功能的基因，同时包含重金属抗性相关基因。|aacA7|blaVIM-2|aacA4|盒式阵列（可介导对碳青霉烯类与氨基糖苷类抗生素的耐药性）位于一个缺陷型Tn402衍生的1类整合子中。该转座子处于一段长度为50273 bp的区域内，该区域两端带有Tn3家族38 bp反向重复序列，并以5 bp正向重复序列（DR）作为侧翼序列；该区域还包含额外的转座子片段、5个插入序列以及一个Tn3衍生反向重复微型元件（Tn3-Derived Inverted-Repeat Miniature Element）。结合纳米孔与Illumina测序的混合策略，研究人员成功完整解析了设拉子假单胞菌中的一株碳青霉烯酶编码巨型质粒。新型巨型质粒的鉴定为解析基因转移的进化效应以及驱动抗生素抗性产生的选择压力提供了全新研究视角。