Identification of regions of the Escherichia coli chromosome specific for neonatal meningitis-associated strains

Abstract

Specific virulence factors associated with the pathogenesis of Escherichia coli strains causing neonatal meningitis (ECNM), such as the K1 capsular polysaccharide, the S fimbriae, and the Ibe10 protein, have been previously identified. However, some other yet unidentified factors are likely to be involved in the pathogenesis of ECNM. To identify specialized unique DNA regions associated with ECNM virulence, we used the representational difference analysis technique. The genomes of two strains belonging to nonpathogenic phylogenetic group A of the ECOR reference collection were subtracted from E. coli strain C5, isolated from a case of neonatal meningitis. Strain C5 belongs to the phylogenetic group B2 as do the majority of ECNM. We have isolated and mapped 64 DNA fragments which are specific for strain C5 and not found in nonpathogenic strains. Of these clones, 44 were clustered in six distinct regions on the chromosome. The sfa and ibe10 genes were located in regions 2 and 6, respectively. A group of genes (cnf1, hra, hly, and prs) known to be present in a pathogenicity island of the uropathogenic strain E. coli J96 colocalized with region 6. The occurrence of these DNA regions was tested in a set of meningitis-associated strains and in a control group composed of non-meningitis-associated strains belonging to the same B2 group. Regions 1, 3, and 4 were present in 91, 82, and 81%, respectively, of the meningitis strains and in 40, 13, and 47% of the control strains. Together, these data suggest that regions 1, 3, and 4 code for factors associated with the ability of E. coli to invade the meninges of neonates.

FIG. 1

Distribution of specific sequences on the chromosome of E. coli strain RS218. E. coli strain C5-specific clones were used as probes on Southern blots of DNA from strain RS218 digested with the infrequently cutting enzymes BlnI and NotI and were positioned on the linearly represented restriction map (26). The upper arrows indicate the six regions found in this study and delineated by NotI and BlnI fragments with a high density of C5-specific clones. Clones with known homologies are indicated, as are the positions of the probes sfa and ibe10. Region 6 was divided into two subregions according to the mapping of the clones on different XbaI fragments. The lower arrows indicate the 10 segments of the RS218 chromosome in which Rode et al. found additions relative to the E. coli K-12 chromosome. The length (in kilobases) of each addition is indicated in the respective circle. The positions of various virulence factors are also indicated (26). Superscripts to clone names designate the following: 1, TspE4.A7 was positioned by overlapping SauE15.F12; 2, SauE15.F12 also shows reactivity on the plasmid; and 3, TspE4.A8 also shows a weak reactivity on the NotI fragment p.