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Background: The aim of this study was to determine the yearly prevalence and genotype distribution of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae collected over a 3-yr period in Gwangju, Korea., Methods: Clinical isolates of E. coli and K. pneumoniae collected at Chosun University Hospital from September 15, 2005 to September 14, 2008 were evaluated. Antimicrobial susceptibility testing was performed using the Vitek II system (bioMérieux, USA) and agar dilution methods. Screening for ESBL and AmpC β-lactamase genes was performed using PCR amplification of plasmid DNA followed by direct sequencing of the PCR products., Results: The percentage of ESBL-producing isolates was 12.6% (196/1,550) for E. coli and 26.2% (294/1,121) for K. pneumoniae. The ESBL gene sequencing results showed that the most prevalent ESBL types were CTX-M (93.5%) and SHV (12.9%) in E. coli, and SHV (73.2%) and CTX-M (46.3%) in K. pneumoniae. The most common ESBL in E. coli was CTX-M-15-like, followed by CTX-M-14-like, SHV-2a-like, and SHV-12-like. The most prevalent ESBL type in K. pneumoniae was SHV-12, followed by CTX-M-14-like and CTX-M-15-like. Fifty-one percent (21/41) of ESBL-producing K. pneumoniae with ESBL types verified by sequencing also had DHA-1-like AmpC β-lactamases. However, none of the ESBL-producing E. coli was positive in the AmpC β-lactamase PCR analysis., Conclusions: In this study, the most common types of class A ESBLs identified were CTX-M-15-like in E. coli and SHV-12-like in K. pneumoniae.