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

Abstract

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 bla IMP-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. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.