Purpose: To investigate the genomic and plasmid characteristics of a newly discovered Pseudomonas stutzeri strain with a bla_VIM-2-carrying plasmid and novel integron In 1998 isolated from a cerebrospinal fluid specimen in a teaching hospital.
Methods: Species identification was performed by MALDI-TOF MS, and bla_VIM-2 was identified by PCR and Sanger sequencing. Whole-genome sequencing analysis was conducted using the Illumina NovaSeq 6000 and Oxford Nanopore platforms. Integron detection was performed using INTEGRALL. The phylogenetic tree was constructed by using kSNP3.0. Plasmid characteristics were assessed by S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, conjugation experiments, and whole-genome sequencing analysis. Comparative genomics analysis of the plasmid and genetic context of bla_VIM-2 were conducted by using BLAST Ring Image Generator (BRIG) and Easyfig 2.3, respectively.
Results: ZDHY95, an MDR strain of P. stutzeri harboring bla_VIM-2, was identified. It was sensitive only to amikacin and was resistant to carbapenems, β-lactams, aztreonam, fluoroquinolones, and aminoglycosides. Joint S1-PFGE, Southern blot, conjugation assay, and whole-genome sequencing experiments confirmed that the bla_VIM-2 gene was located within class I integron In 1722 of the plasmid and that the surrounding genetic environment was 5ʹCS-aacA4ʹ-30-bla_VIM-2-aacA4ʹ-3ʹCS. The novel class I integron In 1998 was detected on the chromosome of P. stutzeri ZDHY95, and the gene cassette array was 5ʹCS-aacA3-aadA13-cmlA8-bla_OXA-246-arr3-dfrA27-3ʹCS. Phylogenetic analysis showed that antimicrobial resistance gene-carrying P. stutzeri isolates were divided into two clusters, mainly containing isolates from the USA and Pakistan.
Conclusion: A novel bla_VIM-2-carrying conjugative plasmid, pZDHY95-VIM-2, was reported for the first time in P. stutzeri, elucidating the genetic environment and transfer mechanism. The gene structure of the novel class I integron In 1998 was also clarified. We explored the phylogenetic relationship of P. stutzeri with drug resistance genes and suggested that Pseudomonas with metallo-β-lactamases (MBLs) in the hospital environment may cause infection in patients with long-term intubation or after interventional surgery.

Keywords: Pseudomonas stutzeri, bla_VIM-2, In 1998, In 1722, Tn 5563, whole-genome sequencing, bacterial genomics, antibiotic resistance


中文翻译：

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1998 年从脑脊液中分离的新型整合子质粒编码的 VIM-2 生产施氏假单胞菌的出现和遗传表征

目的：研究新发现的施氏假单胞菌菌株的基因组和质粒特征，该菌株具有bla _VIM-2携带质粒和新型整合子，该菌株于1998 年从一家教学医院的脑脊液标本中分离出来。
方法：物种鉴定采用 MALDI-TOF MS 和bla _VIM-2通过 PCR 和 Sanger 测序鉴定。使用 Illumina NovaSeq 6000 和 Oxford Nanopore 平台进行全基因组测序分析。使用INTEGRALL进行整合子检测。系统发育树使用kSNP3.0构建。通过 S1 脉冲场凝胶电泳 (S1-PFGE)、Southern 印迹、缀合实验和全基因组测序分析评估质粒特征。分别使用 BLAST Ring Image Generator (BRIG) 和 Easyfig 2.3对bla _VIM-2的质粒和遗传背景进行比较基因组学分析。
结果： ZDHY95，一种含有bla _VIM-2的P. stutzeri的 MDR 菌株, 被识别。仅对阿米卡星敏感，对碳青霉烯类、β-内酰胺类、氨曲南、氟喹诺酮类和氨基糖苷类耐药。联合S1-PFGE、Southern印迹、偶联分析和全基因组测序实验证实bla _VIM-2基因位于质粒1722的I类整合子内，周围遗传环境为5ʹCS -aacA4ʹ-30-bla _VIM-2 - aacA4ʹ - 3ʹCS。1998年在施氏假单胞菌ZDHY95染色体上检测到新型I类整合子，基因盒阵列为5ʹCS -aacA3-aadA13-cmlA8-bla _OXA-246 - arr3-dfrA27-3ʹCS。系统发育分析表明，携带抗菌素耐药基因的施氏P. stutzeri分离株分为两个簇，主要包含来自美国和巴基斯坦的分离株。
结论：首次报道了一种新的携带bla _{VIM-2的结合质粒}pZDHY95 -VIM-2 ，阐明了其遗传环境和转移机制。新型I类整合子的基因结构在1998年也得到了阐明。我们探讨了P. stutzeri与耐药基因的系统发育关系，并提出假单胞菌属在医院环境中使用金属-β-内酰胺酶（MBLs）可能会导致长期插管或介入手术后的患者感染。

关键词： 施氏假单胞菌，bla _VIM-2，1998年，1722年，Tn 5563，全基因组测序，细菌基因组学，抗生素耐药性