Many multidrug resistant (MDR) bacterial strains are now recognized as belonging to clones that originate in a specific locale, country or even globally. Escherichia coli sequence type 131 (ST131) is one such recently emerged and globally disseminated MDR pandemic clone responsible for community and hospital acquired urinary tract and bloodstream infections. E. coli ST131 was first identified in 2008 as a major clone linked to the spread of the CTX-M-15 extended-spectrum β-lactamase (ESBL)-resistance gene. Since then, E. coli ST131 has also been strongly associated with fluoroquinolone resistance, as well as co-resistance to aminoglycosides and trimethoprim-sulfamethoxazole. Our work has shown that the acquisition of key fitness and virulence determinants by ST131 occurred prior to the development of fluoroquinolone resistance, suggesting their attainment has contributed to the capacity of ST131 to cause human infection. In this seminar, I will discuss our recent work on the molecular characterisation of E. coli ST131, including the use of transposon-directed insertion-site sequencing (TraDIS) to define key virulence features of this globally significant clone.