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The aim of this study was to characterize by a multi-level approach extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eight ESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitals located in the North (A) or Centre (B, C) regions during two time periods (2006–7 and 2010) were analyzed. Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL characterization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis. Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2, -5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local …