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JAC Advance Access originally published online on March 6, 2009
Journal of Antimicrobial Chemotherapy 2009 63(5):901-908; doi:10.1093/jac/dkp061
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© The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

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

Cristina Pellegrini1, Paola Sandra Mercuri2, Giuseppe Celenza1, Moreno Galleni2, Bernardetta Segatore1, Elisa Sacchetti1, Roberto Volpe3, Gianfranco Amicosante1 and Mariagrazia Perilli1,*

1 Dipartimento di Scienze e Tecnologie Biomediche, University of L’Aquila, L’Aquila, Italy 2 Laboratoire de Macromolécules Biologiques, Centre d’Ingénierie des Protéines, Université de Liège, Liège, Belgium 3 Dipartimento di Chimica, Ingegneria Chimica e Materiali, University of L’Aquila, L’Aquila, Italy

Received 25 April 2008; returned 2 July 2008; revised 23 January 2009; accepted 30 January 2009

* Corresponding author. Tel: +39-0862433489; Fax: +39-0862433433; E-mail: perilli{at}univaq.it

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.

Keywords: integrons , metallo-β-lactamases , MBLs


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