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

Abstract

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 In1998 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 In1722 of the plasmid and that the surrounding genetic environment was 5ʹCS-aacA4ʹ-30-bla_VIM-2-aacA4ʹ-3ʹCS. The novel class I integron In1998 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 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.

Keywords:

• Pseudomonas stutzeri
• bla_VIM-2
• In1998
• In1722
• Tn5563
• whole-genome sequencing
• bacterial genomics
• antibiotic resistance

Acknowledgments

The authors are grateful to Dr. Zhe Yin for providing the recipient bacteria PAO1Ri for the conjugation experiment in this paper.

Data Sharing Statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval

The ethical protocol was approved by the Ethics Committee of First Affiliated Hospital of Zhejiang University (no. 2018-752).

Disclosure

The authors report no conflicts of interest in this work.