A Pseudomonas putida strain showing broad-spectrum resistance to β-lactams, including expanded-spectrum cephalosporins and carbapenems, was isolated from a patient with a urinary tract infection at the University Hospital of Varese in northern Italy. The isolate was found to produce metallo-β-lactamase activity and to harbor a 50-kb plasmid, named pVA758, carrying a new bla _IMP determinant, named bla _IMP-12 . Plasmid pVA758 was not self-transferable by conjugation to either Escherichia coli or Pseudomonas aeruginosa but could be introduced by electroporation and maintained in the latter host, where it conferred resistance or decreased susceptibility to various β-lactams. The IMP-12 enzyme is quite divergent from other IMP variants: its closest relatives are IMP-8 and IMP-2 (89 and 88% sequence identity, respectively), and IMP-1 is 85% identical to IMP-12. The bla _IMP-12 determinant is carried on an integron-borne gene cassette whose attC recombination site is related to those present in cassettes containing bla _IMP-1 , bla _IMP-6 , bla _IMP-7 , bla _IMP-10 , and bla _IMP-11 and unrelated to that present in cassettes containing bla _IMP-2 and bla _IMP-8 . IMP-12 was overproduced in E. coli by using a T7-based expression system and was purified by cation-exchange chromatography followed by gel filtration. Kinetic analysis revealed that, like other IMP variants, IMP-12 exhibits an overall preference for cephalosporins and carbapenems rather than for penicillins and does not hydrolyze temocillin and aztreonam. However, IMP-12 also exhibits some notable functional differences from other IMP variants, including uniformly poor activity toward penicillins ( k _cat / K _m values, around 10 ⁴ M ⁻¹ · s ⁻¹ ) and a remarkably high K _m (around 900 μM) for imipenem.

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