Identification of blaIMP-22 in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location

• Published in: Journal of antimicrobial chemotherapy 2009-03, Vol.63 (5), p.901-908
• Main Authors: Pellegrini, Cristina, Mercuri, Paola Sandra, Celenza, Giuseppe, Galleni, Moreno, Segatore, Bernardetta, Sacchetti, Elisa, Volpe, Roberto, Amicosante, Gianfranco, Perilli, Mariagrazia
• Format: Article
• Language: English
• Subjects: Antibiotics. Antiinfectious agents. Antiparasitic agents; Biological and medical sciences; General aspects; Human infectious diseases. Experimental studies and models; Infectious diseases; integrons; MBLs; Medical sciences; metallo-β-lactamases; Pharmacology. Drug treatments
• ISSN: 0305-7453; 1460-2091
• DOI: 10.1093/jac/dkp061

Objectives The aim of the study was the biochemical characterization of a new variant of the metallo-β-lactamase, IMP-22. Moreover, the genetic environment of the blaIMP-22 gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L’Aquila, Italy. Methods Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/IMP-22. Steady-state kinetic parameters (Km and Vmax) were determined for a large pattern of substrates. Results A new IMP metallo-β-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The blaIMP-22 encodes for a pre-protein of 246 amino acids and the N-terminus of the mature β-lactamase (NH2-PDLK) was also determined. The molecular mass and pI were 24 930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (Km and Vmax), IMP-22 was found to hydrolyse narrow- and extended-spectrum β-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC50 of 800, 750 and 300 µM, respectively. Conclusions The finding of the blaIMP-22 gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of blaMBL genes in several bacterial species and in different environments.