We conducted a retrospective study to investigate the epidemiology of Enterobacteriaceae producing extended-spectrum beta-lactamase (ESBLE) in our hospital. We determined the occurrence of extended-spectrum beta-lactamase (ESBL) in Enterobacteriaceae over a 2-year period. We also characterised ESBLs by isoelectric focusing (IEF) and investigated the epidemiological relatedness of EBLSE by pulsed field gel electrophoresis (PFGE). During this period, 70 patients were colonised/infected with one or several strains of EBLSE, giving a crude incidence of 0.095 per 1000 patient-days. We found that ESBL-producing Enterobacter aerogenes were the main source of ESBLE dissemination. Indeed, 59.5% of ESBLE were E. aerogenes and 21.9% of the other ESBLE resulted from a plasmid transfer originating from E. aerogenes. IEF and PFGE analysis demonstrated that the dissemination of ESBL from E. aerogenes in our hospital was due to a single clone that always harbours TEM-24. This emphasises the importance of standard contact isolation precautions and the early detection of ESBLE-colonised patients in high risk departments like intensive care units.