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Despite recent advances in molecular epidemiology and pathogenecity analyses of extraintestinal Escherichia coli infections, detailed analyses identifying virulence factors of E. coli isolates from pediatric urosepsis patients have not been reported. This study was conducted to explore and differentiate bacterial virulence factors associated with urosepsis and 2 other severe parenchymal infections, acute pyelonephritis (APN) and acute lobar nephronia (ALN), in pediatric patients.Patients included in this study were those who fulfilled the diagnostic criteria of urosepsis, APN, and ALN, without underlying disease or structural anomalies, excluding those with vesicoureteral reflux. Patients with cystitis were included as controls. E. coli isolates from urine (cystitis, APN, and ALN) or blood (urosepsis) specimens were analyzed using polymerase chain reaction (PCR) for 25 virulence genes.A total of 147 children (24 cystitis, 45 APN, 48 ALN, and 30 urosepsis) were enrolled in the study. Distinct syndrome-specific differences in the distribution for certain virulence genes, but conservation across syndromes for others, were found. In addition, urosepsis isolates presented higher aggregate virulence factor scores (P < 0.0001) compared with cystitis, APN, and ALN isolates. By contrast, cystitis isolates showed significantly lower aggregate virulence factor scores than all 3 invasive urinary bacterial infections; APN (P < 0.01), ALN (P < 0.01), and urosepsis (P < 0.0001).Our findings suggested that urosepsis isolates carry more virulence factors and are likely more urovirulent compared with cystitis, APN, and ALN isolates.