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In the early 2000s, there was a rapid increase in extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in hospital settings throughout Japan. The reasons for this rapid increase are unclear.Between 2002 and 2003, 142 clinical isolates of E. coli suspected of producing ESBL were obtained from 37 hospitals and commercial clinical laboratories in geographically distinct regions throughout Japan. They were tested for ESBL types and further subtyped for serogroups, fimH single nucleotide polymorphism, pulsed-field gel electrophoresis patterns and multilocus sequence type (MLST). Representative isolates were also subjected to plasmid analysis.Of 142 E. coli isolates suspected of producing ESBL, 130 were confirmed as harbouring blaCTX-M by PCR analysis and sequencing. Of these, 84 (65%) harboured CTX-M-9-group blaCTX-M. Two serogroups O25 and O86 accounted for 41% of the 130 blaCTX-M-positive E. coli. All O86 serogroup strains belonged to ST38 by MLST and they formed 18% of all the blaCTX-M-positive E. coli. Serogroup O25 strains belonged to ST131 and ST73, and formed 21% and 1% of blaCTX-M-positive E. coli, respectively. Seven characterized plasmids carrying blaCTX-M genes belonged to three distinct incompatibility groups: IncF, IncN and IncI1.In this study, clonally related strains of E. coli accounted for a large proportion of blaCTX-M-positive E. coli. This high proportion of clonal groups identified in different regions of Japan suggests their recent spread by mechanisms other than healthcare-associated transmission. These observations imply that restricting antimicrobial use in human clinical settings may have limited impact on the spread of ESBL-producing E. coli.