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Urinary tract infections (UTI) are common in children and contribute significantly to morbidity and mortality. The major cause is Escherichia coli, carrying multiple virulence-associated factors (VFs). However, the specific traits that distinguish childhood uropathogenic E. coli from fecal commensals of healthy children are incompletely defined.We used a multiplex polymerase chain reaction–based reverse line blot assay, several additional polymerase chain reactions and phenotypic methods to compare distributions of virulence traits (22 VF genes, UTI-associated O types, phylogenetic groups, sequence type 131 and expression of selected VFs), between 212 E. coli isolates from children ≤5 years with UTI (109 cystitis and 103 pyelonephritis) and 115 fecal isolates from healthy children of similar age, collected during the same time period.The studied traits were most prevalent among pyelonephritis, followed closely by cystitis isolates and were uncommon among fecal isolates. Eight VF genes differentiated pyelonephritis from cystitis isolates, but aggregate VF scores in these 2 UTI groups were similar. Most of the studied phenotypic characteristics showed a similar descending prevalence gradient from pyelonephritis, through cystitis, to fecal isolates. Coexpression of biofilm components, curli and cellulose, was strongly associated with pyelonephritis, phylogenetic group B2, individual VF genes and higher VF scores. Two-thirds (67%) of clinical isolates belonged to phylogenetic group B2 and, of these, 12% belonged to the sequence type 131 clonal group, compared with 14% and 1%, respectively, of fecal isolates.These findings identify specific virulence factors, O types and a virulent clonal group (sequence type 131), as potential targets for UTI prevention strategies in children.