OBJECTIVES: To investigate the presence of extended-spectrum beta-lactamases (ESBLs) among Pseudomonas aeruginosa clinical isolates referred to two Belgian reference laboratories. METHODS: Antibiograms were analysed for P. aeruginosa isolates referred between 2004 and 2008. Isolates resistant to ceftazidime (MIC > 8 mg/L) and with a positive double-disc synergy test between ceftazidime and clavulanate were serotyped and screened for the presence of ESBL-encoding genes. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought by PCR in ESBL-producing isolates with positive imipenem/EDTA synergy tests. PFGE of SpeI-digested genomic DNA was used to compare isolates and selected strains were characterized by multilocus sequence typing. RESULTS: Forty-eight (2.2%) of 2150 P. aeruginosa isolates were confirmed as class A ESBL-producing isolates by molecular testing. bla(BEL) and bla(PER) alleles were detected, respectively, in 39 and 10 P. aeruginosa isolates originating from 16 hospitals (two isolates were simultaneously positive for BEL and PER). Fifteen of the isolates were found to co-produce ESBLs and VIM carbapenemases. These strains were pan-resistant and remained susceptible only to colistin (MICs <or= 2 mg/L). The majority of the ESBL-producing isolates belonged to the same PFGE clone and were identified as ST235; serotype O11. CONCLUSIONS: BEL enzymes were produced by 80% of P. aeruginosa isolates with phenotypic evidence of ESBL production. BEL or PER ESBLs co-existed with VIM carbapenemases in 15 isolates and caused outbreaks in four hospitals. Our data further highlight the epidemic potential of the international clone ST235, which may have acquired bla(BEL-1) gene cassettes from a yet unidentified local gene reservoir.