Isolation of unique DNA sequences from uropathogenic Escherichia coli using a subtraction hybridization tecnique

Most Escherichia coli isolated from urinary tract infections appear to belong to highly defined clones and have a high incidence of virulence-associated factors. Genes for hemolysin production (hly) and pyelonephritis-associated-pili (pap) have been isolated and their incidence found to correlate with severity of infection. The highest incidence of these genes is found in pyelonephritis isolates, and a somewhat lower incidence is found in cystitis isolates. However, a significant proportion of cystitis isolates are hly¯ and pap¯. This finding has led to a question about the possible occurrence of other genetically determined cystitis virulence-associated factors. If such genes exist, their isolation could help increase the understanding of E. coli pathogenicity as well as provide a useful clinical tool. The goal of the studies reported here, was isolated unique sequences of DNA from a single cystitis isolate that encode for hly sequences and unique cystitis sequences. To accomplish this a subtraction hybridization method was employed in which chromosomal DNA from a cystitis isolate (A16) was hybridized in a solution with excess biotinylated DNA from a non-pathogenic fecal isolate (J198). After hybridization avidin was added and the solution was chromatographed over cupric-charged agarose. This matrix selectively binds avidin and avidin-DNA complexes. The effluent should contain only non-biotinylated DNA, enriched for unique A16 sequences which have no homology with J198 sequences. The results of these define critical points in the subtraction hybridization method. They demonstrate that biotinylated DNA in more hydrophobic than non-biotinylated DNA and that it has an increased tendency for non-specific absorption. Several methods are evaluated to increase the recovery of biotin-DNA. Additionally, these studies illustrated the importance of using a hybridization buffer that does not have substances (e.g., formamide and SDS) that interfere with cupric-chelate chromatography. These studies also suggest that the subtraction hybridization method can be used to enrich chromosomal DNA. Further work is required to isolate unique sequences of DNA from a cystitis isolate and to evaluate their incidence in virulent and non-virulent E. coli strains.