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We investigated Escherichia coli populations in a metropolitan river after an extreme flood event. Between nine and 15 of the 23 selected sites along the river were sampled fortnightly over three rounds. In all, 307 E. coli were typed using the PhP typing method and were grouped into common (C) or single (S) biochemical phenotypes (BPTs). A representative from each of the 31 identified C-BPTs was tested for 58 virulence genes (VGs) associated with intestinal and extra-intestinal E. coli, resistance to 22 antibiotics, production of biofilm and cytotoxicity to Vero cells. The number of E. coli in the first sampling round was significantly (P < 0.01) higher than subsequent rounds, whereas the number of VGs was significantly (P < 0.05) higher in isolates from the last sampling round when compared to previous rounds. Comparison of the C-BPTs with an existing database from wastewater treatment plants (WWTPs) in the same catchment showed that 40.6% of the river isolates were identical to the WWTP isolates. The relatively high number of VGs and antibiotic resistance among the C-BPTs suggests possessing and retaining these genes may provide niche advantages for those naturalised and/or persistent E. coli populations which may pose a health risk to the community.