Emergence of a novel transferable megaplasmid driving blaVIM-24 and tmexCD3-toprJ3 dissemination in clinical Pseudomonas fulva isolates.

To investigate the genetic characteristics and transmission mechanism of clinical Pseudomonasfulva isolates with transferable megaplasmid co-carrying blaVIM-24 and tmexCD3-toprJ3. Bacterial identification was performed using MALDI-TOF/MS, and antimicrobial susceptibility testing was carried out using agar dilution and broth microdilution. The genetic context of drug resistance genes and plasmid characteristics was analyzed by S1-PFGE, Southern blotting, conjugation experiments, and whole-genome sequencing analysis. Comparative genomics analysis of the plasmids and genetic context was conducted by using BLAST Ring Image Generator (BRIG) and Easyfig 2.3. Phylogenetic analysis of P. fulva strains and pJBCL41-like megaplasmids was performed by Snipy and Mega, respectively. Clinical P. fulva strains, ZDHY316 and ZDHY414, with transferable megaplasmids co-carrying blaVIM-24 and tmexCD3-toprJ3. The megaplasmids pVIM-24-ZDHY316 and pVIM-24-ZDHY414 carry multiple drug-resistant genes and integrate numerous integrons and transposon truncations from different origins. ΔTn6855-ΔTn6758 is the new discoovered co-transfer module carrying nfxB-mexCD-oprJ, which only exists in chromosomes and megaplasmids. Phylogenetic analysis of pJBCL41-like megaplasmids showed their evolution towards carrying more drug-resistance genes and mobile genetic elements. Additionally, ZDHY316 has another transferable plasmid, pVIM-1-ZDHY316, which carries the novel integron In2008 with the GCA of 5'CS- blaVIM-1-aac(6')-Ib-3'CS. Phylogenetic analysis of P. fulva strains showed that China is the country with the most P. fulva isolated clinically, with strains prevalent and evolving in hospitals. The mosaic structure of the megaplasmid, characterized by integrons and transposons, underscores its role in resistance gene dissemination and highlights the adaptability of non-standard pathogens like P. fulva. The horizontal transfer potential of this megaplasmid poses a significant challenge to clinical infection control. Enhanced surveillance of non-standard pathogens and their plasmids is essential.