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More than a quarter of women who experience acute cystitis develop recurrence but information on specific urovirulent genetic profile of uropathogenic Escherichia coli associated with recurrent cystitis is still limited. In this prospective cohort study, index episode E. coli from a cohort of 46 sexually active women with acute cystitis who reported recurrence during followup were grouped into repeat infection (RI) and single infection (SI) isolates, based on enterobacterial repetitive intergenic consensus (ERIC) PCR profile comparison with subsequent E. coli isolated from same women. PCR for phylogrouping and 15 virulence genes along with test for biofilm formation were done. Virulence score was calculated for each isolate as number of virulence genes detected. Among 46 index E. coli, 22 were RI, and 24 were SI isolates. RI isolates had phylogroup B2 as majority (54.5%) which is typically described as more virulent phylogroup and virulence score for RI isolates was also significantly higher compared to SI isolates. Virulence gene malX (p = 0.03) was significantly associated with RI isolates. 68.2% RI isolates were strong to moderate biofilm producers in comparison to 33.3% SI isolates, an important survival strategy to reside in bladder and or vagina. Overall, E. coli associated with recurrent cystitis appear to be more virulent and malX seems to have a role in causing repeat infection.