[en] 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 blaIMP determinant, named blaIMP-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 blaIMP-12 determinant is carried on an integron-borne gene cassette whose attC recombination site is related to those present in cassettes containing blaIMP-1, blaIMP-6, blaIMP-7, blaIMP-10, and blaIMP-11 and unrelated to that present in cassettes containing blaIMP-2 and blaIMP-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 (kcat/Km values, around 104 M-1 · s-1) and a remarkably high Km (around 900 μM) for imipenem.
Docquier, Jean-Denis ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Riccio, M. L.; Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, I-53100 Siena, Italy
Mugnaioli, C.; Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, I-53100 Siena, Italy
Luzzaro, F.; Laboratorio di Microbiologia, Ospedale di Circolo, Università dell'Insubria, I-21100 Varese, Italy
Endimiani, A.; Laboratorio di Microbiologia, Ospedale di Circolo, Università dell'Insubria, I-21100 Varese, Italy
Toniolo, A.; Laboratorio di Microbiologia, Ospedale di Circolo, Università dell'Insubria, I-21100 Varese, Italy
Amicosante, G.; Dipto. di Sci./Tecnologie Biomediche, Università di L'Aquila, I-67100 L'Aquila, Italy
Rossolini, G. M.; Dipartimento di Biologia Molecolare, Sezione di Microbiologia, Università di Siena, I-53100 Siena, Italy, Dipartimento di Biologia Molecolare, Università di Siena, Policlinico Le Scotte, 53100 Siena, Italy
Language :
English
Title :
IMP-12, a new plasmid-encoded metallo-β-lactamase from a Pseudomonas putida clinical isolate
Publication date :
2003
Journal title :
Antimicrobial Agents and Chemotherapy
ISSN :
0066-4804
eISSN :
1098-6596
Publisher :
American Society for Microbiology, Washington, United States - District of Columbia
Volume :
47
Issue :
5
Pages :
1522-1528
Peer reviewed :
Peer Reviewed verified by ORBi
Name of the research project :
European Training and Mobility of Researchers Network on Metallo-beta-Lactaqmases (grant no. FMRX-CT98-0232)
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