Pseudomonas aeruginosa remains one of the most important pathogens in the nosocomial setting [1]. P. aeruginosa exhibits intrinsic resistance to several antimicrobial agents. The antipseudomonal β-lactams represent a major weapon against Pseudomonas infections, either for monotherapy or for combination therapy, for which β-lactams almost invariably represent one of the components. Therefore, resistance to these agents constitutes a major challenge for anti-Pseudomonas chemotherapy. Several mechanisms can contribute to β-lactam resistance in P. aeruginosa, including β-lactamase production, outer membrane impermeability and active efflux mediated by RND-type efflux systems [1]. During the last decade, the metallo-β-lactamases (MBLs) have emerged as new threatening mechanisms of broad-spectrum β-lactam resistance in P. aeruginosa. In fact, these enzymes can efficiently degrade virtually all antipseudomonal β-lactams (except aztreonam), while they are not susceptible to therapeutic β-lactamases inhibitors [2]. Based on amino acid sequence homology, these MBLs have been classified into four major types: IMP, VIM, SPM and GIM. Clinical isolates harbouring the MBLs IMP and VIM have been increasingly reported worldwide, mostly in European and Asian countries [2]. This increase in occurrence, types and rate of dissemination of MBLs makes early detection very critical. The benefits of such treatment include the timely implementation of strict infection control practices, as well as clinical guidance. The aim of this study was to determine the presence of these enzymes in imipenem-resistant P. aeruginosa isolates collected at the Centro Hospitalar of Coimbra (CHC), during a two-year period (April 2003 to April 2005) and to ascertain their clonal relationship. CHC is a cluster formed by a central hospital and several specialised units located at distances of less than 8 km, namely, Hospital dos Covoes (central hospital), Instituto Maternal (maternity hospital) and Hospital Pediatrico (paediatric hospital). The Microbiology Laboratory also analysed samples from another hospital (Hospital de Pombal) located 30 km away. Bacterial identification and minimal inhibitory concentrations (MIC) were performed with the MicroScan WalkAway (Dadebehring) system according to the instructions of the manufacturer and API32GN or API PSE systems (bioMerieux). All intermediate strains were considered as nonsusceptible strains. The MICs of β-lactams were also determined by E-test in isolates that presented the enzyme VIM-2. The results were interpreted on the basis of CLSI recommended breakpoints [3]. The screening of MBLs was done by the double combined disk test [4]. Polymerase chain reaction (PCR) analyses for the detection of MBLs genes (blaIMP, blaVIM, blaGIM and blaSPM-1) were carried out for all strains in which the screening test gave positive results [5]. The amplicons were directly sequenced on both DNA strands on an ABI PRISM 377 automated sequencer. The nucleotide and deduced amino acid sequences were analysed with software available online (http://blast.ncbi.nlm.nih.gov/ Eur J Clin Microbiol Infect Dis (2008) 27:1269–1271 DOI 10.1007/s10096-008-0579-2