Characterization of two novel VIM-type metallo-β-lactamases, VIM-84 and VIM-85, associated with the spread of IncP-2 megaplasmids in Pseudomonas aeruginosa.

This study aimed to characterize two novel VIM-type metallo-β-lactamases, VIM-84 and VIM-85, and reveal the important role of the IncP-2 type megaplasmids in the spread of antimicrobial resistance (AMR) genes. VIM-84 and VIM-85 were encoded by two novel genes bla VIM-84 and bla VIM-85 which showed similarity to bla VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. The bla VIM-85 gene was identified in a megaplasmid, which was related to 17 megaplasmid sequences with sizes larger than 430 kb, deposited previously in Genbank. A comparative analysis of complete plasmid sequences showed highly similar backbone regions and various AMR genes. A phylogenetic tree revealed that these megaplasmids, which were widely distributed globally, were vehicles for the spread of AMR genes. The bla VIM-24, bla VIM-84, and bla VIM-85 genes were cloned into pGK1900, and the recombinant vectors were further transformed into Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. The antimicrobial susceptibility test of the cloning strains showed high levels of resistance to β-lactams while they remained susceptible to aztreonam. Enzymatic tests revealed that both, VIM-84 and VIM-85, exhibited higher activity in hydrolyzing β-lactams compared to VIM-24. A D117N mutation found in VIM-24 affected binding to the antibiotics. IMPORTANCE The metallo-β-lactamases-producing Pseudomonas aeruginosa strains play an important role in hospital outbreaks and the VIM-type enzyme is the most prevalent in European countries. Two novel VIM-type enzymes in our study, VIM-84 and VIM-85, have higher levels of resistance to β-lactams and greater hydrolytic activities for most β-lactams compared with VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. Notably, the genes bla VIM-85 are carried by three different IncP-2-type megaplasmids which are distributed locally and appear responsible for the spread of antimicrobial resistance genes in hospital settings.