Genomic analysis of a pathogenicity island in uropathogenic Escherichia coli CFT073: distribution of homologous sequences among isolates from patients with pyelonephritis, cystitis, and Catheter-associated bacteriuria and from fecal samples

Abstract

Urinary tract infection is the most frequently diagnosed kidney and urologic disease and Escherichia coli is by far the most common etiologic agent. Uropathogenic strains have been shown to contain blocks of DNA termed pathogenicity islands (PAIs) which contribute to their virulence. We have defined one of these regions of DNA within the chromosome of a highly virulent E. coli strain, CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. The 57,988-bp stretch of DNA has characteristics which define PAIs, including a size greater than 30 kb, the presence of insertion sequences, distinct segmentation of K-12 and J96 origin, GC content (42.9%) different from that of total genomic DNA (50.8%), and the presence of virulence genes (hly and pap). Within this region, we have identified 44 open reading frames; of these 44, 10 are homologous to entries in the complete K-12 genome sequence, 4 are nearly identical to the sequences of E. coli J96 encoding the HlyA hemolysin, 11 encode P fimbriae, and 19 show no homology to J96 or K-12 entries. To determine whether sequences found within the junctions of the PAI of CFT073 were common to other uropathogenic strains of E. coli, 11 probes were isolated along the length of the PAI and were hybridized to dot blots of genomic DNA isolated from clinical isolates (67 from patients with acute pyelonephritis, 38 from patients with cystitis, 49 from patients with catheter-associated bacteriuria, and 27 from fecal samples). These sequences were found significantly more often in strains associated with the clinical syndromes of acute pyelonephritis (79%) and cystitis (82%) than in those associated with catheter-associated bacteriuria (58%) and in fecal strains (22%) (P < 0.001). From these regions, we have identified a putative iron transport system and genes other than hly and pap that may contribute to the virulent phenotype of uropathogenic E. coli strains.

FIG. 1

Features of the pathogenicity island of E. coli CFT073. ORFs defined by nucleotide sequencing are shown as arrows. The ORF designations, below the arrows, are defined in Table 2. The direction of each arrow indicates the predicted direction of transcription. The top line of shaded boxes indicates that ORFs in this region are highly homologous to or identical with ORFs previously identified in E. coli K-12 (dark shading) (2) or uropathogenic J96 (light shading) (1, 8, 20, 21, 29, 30, 32, 40). The unshaded boxes on the second line indicate regions in which the complete nucleotide sequence was obtained in both directions. Sample sequencing was conducted only in areas not covered by unshaded boxes; these regions matched K-12 or J96 sequences with respect to restriction endonuclease sites or nucleotide sequence identity. The positions of restriction fragments or PCR products that were used as probes for DNA hybridization are shown below the ORFs. The scale at the bottom is shown in kilobases. Cosmid clone 8-3f includes ∼35 kb of the PAI beginning 3 kb to the left of the depicted left junction (0 kb) and extending to the center of the pap operon (∼39 kb). Subclone 8HS9 includes PAI sequences from ∼10 to 17 kb. Cosmid clone 5-4a includes ∼37 kb extending from the center of the hemolysin gene cluster (∼23.5 kb) to 5 kb to the right of the right junction (58 kb). Subclone 5H11B extends from ∼38 to 49 kb.