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.

Co-Existence of KPC-2, LAP-2, and CTX-M-65 in an ST1469 Multidrug-Resistant Klebsiella pneumoniae Strain in China

“…To verify the transferability of the bla KPC-2 -bearing plasmid, conjugation experiments were conducted using rifampin-resistant P. aeruginosa PAO1Ri as recipients, as described previously. 15 The transconjugants were selected on Mueller-Hinton agar plates (OXOID, Hampshire, United Kingdom), containing 200 mg/L rifampicin and 1 mg/L meropenem. The AST results of transconjugant SJ25-P indicated that the plasmid pSJ25_KPC_CTX was successfully transferred into the recipient and impacted bacterial resistance ( Table 1 ).…”

Uncovering bacterial hosts of class 1 integrons in an urban coastal aquatic environment with a single-cell fusion-polymerase chain reaction technology

“…Previously, blaOXA 10 was referred to as pse-2 (Pseudomonas-specific enzymes) due to its initial discovery in P. aeruginosa plasmids. Class 1 integron-associated blaOXA 10 gene cassettes have been found in a variety of Gammaproteobacteria pathogens, including those from the Acinetobacter, Pseudomonas, Gallibacterium, Providencia, and Stutzerimonas genera [71][72][73][74][75] . Through epicPCR, we associated the blaOXA 10 gene cassette with a Moraxellaceae-family host (Figure 2D).…”

First report of Klebsiella pneumoniae co-producing OXA-181, CTX-M-55, and MCR-8 isolated from the patient with bacteremia

“…The susceptibility of K. pneumoniae 5589 and its transconjugants to antibiotics was tested using the agar dilution method, except for the polymyxins, which was performed using the broth microdilution method (Liu et al, 2021). The results were interpreted based on the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines.…”

“…The locations of plasmids harboring the bla OXA-181 and mcr-8 were determined by Southern blotting and hybridization with digoxigenin-labeled specific probes. Furthermore, rifampin-resistant P. aeruginosa PAO1Ri was used as a recipient bacterium in transformation conjugation experiments to investigate whether the plasmids can transfer (Liu et al, 2021). The transconjugants which showed growth on Mueller-Hinton medium simultaneously containing 300 mg/L rifampicin and 2 mg/L meropenem were identified by MALDI-TOF/MS.…”