Emergence and Genetic Characterization of Plasmid-Encoded VIM-2-Producing Pseudomonas stutzeri with Novel Integron In1998 Isolated from Cerebrospinal Fluid.

To investigate the genomic and plasmid characteristics of a newly discovered Pseudomonas stutzeri strain with a bla VIM-2-carrying plasmid and novel integron In1998 isolated from a cerebrospinal fluid specimen in a teaching hospital. 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. 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 In1722 of the plasmid and that the surrounding genetic environment was 5'CS-aacA4'-30-bla VIM-2-aacA4'-3'CS. The novel class I integron In1998 was detected on the chromosome of P. stutzeri ZDHY95, and the gene cassette array was 5'CS-aacA3-aadA13-cmlA8-bla OXA-246-arr3-dfrA27-3'CS. Phylogenetic analysis showed that antimicrobial resistance gene-carrying P. stutzeri isolates were divided into two clusters, mainly containing isolates from the USA and Pakistan. 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 In1998 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.