The genetic characteristics and molecular epidemiology of extended-spectrum -lactamases (ESBLs) among Escherichia coli isolates were investigated at a general hospital and its associated health care facilities in Stockholm, Sweden, during the period from 2001 to 2006. Of 87 consecutive nonduplicate ESBL-positive isolates, 80 isolates encoded CTX-M-type ESBLs, 64 of which were group 1 enzymes. TEM-type and OXA-type -lactamases were encoded in 63 and 59% of the ESBL isolates, respectively. Pulsed-field gel electrophoresis (PFGE) analysis revealed 40 different pulsotypes, consisting of 11 clones accounting for 66% of all isolates, and 29 unique patterns. Moreover, of the 11 clones, clones 1 and 4 comprised half of the clonally related isolates (28 of 57). Clone 1 was a persistent endemic clone in the area throughout the years, and clone 4 emerged in 2003. However, in recent years, clone 1 isolates were no longer predominant and were gradually replaced by new emerging strains. Concerning -lactamase gene profiles in relation to PFGE pulsotypes, clone-related bla profiles were observed in certain clones, while in most cases different bla profiles could be observed in the same clone, and the same bla profile could be present in different clones. The molecular epidemiology of ESBLpositive E. coli in the area shows shifts in predominant strains and increased clonal diversity over time. The study also indicated that both clonal spread of epidemic strains and transfer of transposable genetic elements might contribute to the proliferation of ESBLs.Extended-spectrum -lactamases (ESBLs) are the major cause of resistance to oxyimino-cephalosporins in Enterobacteriaceae (4). ESBLs are mostly plasmid-mediated bacterial enzymes that are able to hydrolyze a wide variety of penicillins and cephalosporins. Most ESBLs have evolved by genetic mutation from native -lactamases, particularly TEM-1, TEM-2, and SHV-1. These parent enzymes are commonly found in gram-negative bacteria, particularly in Enterobacteriaceae (4). Until the 2000s, most of the ESBLs were structurally related to the narrow-spectrum TEM-and SHV-type -lactamases, with one to several amino acid substitutions surrounding their active site (4). The genetic mutations that give rise to ESBLs broaden the parental resistance pattern to a phenotype that includes resistance to broad-spectrum cephalosporins (e.g., cefotaxime [CTX] and ceftazidime [CAZ]) and monobactams (e.g., aztreonam) (9). Furthermore, in the late 1990s, a novel type of ESBLs, the CTX-M enzymes, emerged worldwide, mostly from Escherichia coli (2, 4). The more than 50 CTX-M enzymes thus far reported may be grouped into five main subgroups according to amino acid sequence similarity (CTX-M-1, CTM-M-2, CTX-M-8, CTX-M-9, and CTX-M-25) (2). Most of the CTX-Ms hydrolyze CTX better than CAZ. However, several CTX-Ms, including CTX-M-15, which is now the most widespread CTX-M enzyme worldwide (5, 10, 13, 21, 23), also hydrolyze CAZ efficiently (17). The OXA-type enzymes are another growing family of ESBLs and ar...