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This study aimed to assess the prevalence and associated risk factors for extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EK) acquisition among patients staying in medical and surgical Intensive Care Units (ICU) in Northern Thailand. Rectal swabs were collected from 206 ICU patients upon admission and discharge. Overall, the ESBL-EK acquisition rate among patients during ICU stay was 29.6%. Acquisition rate was significantly higher for K. pneumoniae (20.9%) than that of E. coli (12.1%) (p = 0