IMP-12, a new plasmid-encoded metallo-beta-lactamase from a Pseudomonas putida clinical isolate.
A Pseudomonas putida strain showing broad-spectrum resistance to beta-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-beta-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 beta-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 micro M) for imipenem.
Docquier
Jean-Denis
JD
Dipartimento di Biologia Molecolare, Sezione di Microbiologia Università di Siena, I-53100 Siena, Italy.
Riccio
Maria Letizia
ML
Mugnaioli
Claudia
C
Luzzaro
Francesco
F
Endimiani
Andrea
A
Toniolo
Antonio
A
Amicosante
Gianfranco
G
Rossolini
Gian Maria
GM
D000595:Amino Acid Sequence; D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D001618:beta-Lactamases