PCR versus Hybridization for Detecting Virulence Genes of Enterohemorrhagic Escherichia coli

We compared PCR amplification of 9 enterohemorrhagic Escherichia coli virulence factors among 40 isolates (21 O/H antigenicity classes) with DNA hybridization. Both methods showed 100% of the chromosomal and phage genes: eae, stx, and stx2. PCR did not detect 4%–20% of hybridizable plasmid genes: hlyA, katP, espP, toxB, open reading frame (ORF) 1, and ORF2.


PCR versus
E nterohemorrhagic Escherichia coli (EHEC) patho- genicity is usually linked to a Shiga toxin (1,2) and virulence factors, including adhesins, toxins, invasins, protein secretion systems, iron uptake systems, and several unidentifi ed functions (3,4), which are unrelated to strain phylogeny.In many laboratories, sorbitol-MacConkey medium is commonly used to screen for the slow sorbitol fermentation phenotype of the most common Shiga toxin-containing strain: O157:H7 (5), but this process does not address the pathogenic potential of the remaining sorbitol-positive E. coli.These organisms can be detected by immunologic methods or PCR evaluation of virulence factors.PCR is the most useful method for virulence factor detection, and others have made convincing arguments for its use in characterizing the virulence factor patterns of potential pathogens (6,7).
Variation in virulence factor targets and use of different PCR primers contribute to variable results in detecting the most common virulence factors: stx1, stx2, eae, and hlyA (or ehxA).Variation in amplifi cation success is likely to increase because more virulence factor variants are certain to emerge as more EHEC and Shiga toxin-producing E. coli (STEC) strains are identifi ed.This study addresses the potential for a broad and well-characterized set of control strains relative to virulence factor amplifi cation and confi rmed by Southern hybridization.
Probe hybridization was carried out in rotating hybridization bottles (Fisher Scientifi c Isotemp hybridization oven, Fisher Biotech) in 20 mL 6× SSC, 1% SDS at Seventy-fi ve percent (30/40) of the pathogenic E. coli strains tested contained at least 1 stx gene, 23% (9/40) were positive for both stx1 and stx2.The most common gene detected was intimin (eae), which was positive by both PCR and hybridization in 37/40 (93%) of the strains.While eae is strongly correlated with Shiga toxin, the adherence phenotype conveyed may be suffi cient to cause a pathogenic state because 4 of the clinical isolates investigated contained only the eae gene.

Conclusions
The current accepted standard for EHEC identifi cation is amplifi cation of stx1, stx2, eae, and hlyA by PCR.However, this technology is generally only available at large hospital or state health laboratories.Hybridization is superior to culture screening methods and largely complimentary to PCR, but has a potentially broader epidemiologic application since it is unaffected by minor sequence variations that can completely inhibit PCR.
Only 3% of the 360 virulence factor hybridizations made in this study did not amplify.PCR failure is expected with its relatively higher sensitivity to single base primer-hybrid mismatch compared to whole amplicon hybridization.Notably, however, all 12 variations detected were among plasmid-associated virulence factors: 95% (228/240) of the plasmid hybridizable targets were amplifi ed, compared to 100% (120/120) of the hybridizable chromosomal targets.
Although we detected the variable presence of genes ostensibly associated with 2 plasmids (pO157 and pO-SAK1) and the bacterial chromosome, we did not attempt to verify either plasmid or chromosomal locations for any of the amplicons or DNA:DNA hybrids.While all virulence factor targets summarized in this study are subject to change there have been reports of any of the putative chromosomal or plasmid virulence factor targets in this study being found elsewhere.
Prager et al. (7) recently reported, using PCR alone, a wide variety of 25 virulence factor combinations among 266 pathogenic E. coli isolates representing 81 serotypes.Such diversity speaks directly to the need to accurately assess virulence factor presence to evaluate epidemiologic and clinical correlations.A similar 5% failure of the plasmid-associated virulence factor amplifi cations could have implications in such virulence factor correlations.Overall, however, these results are very similar to those of this study of prospective control strains.The use of a single control, such as EDL 933, will inherently bias PCR detection schemes since a failure of amplifi cation in a test will be read as the absence of virulence factor element because it was amplifi able in the control.
If amplifi cation failure is a measure of template variation, we fi nd a much greater variability among plasmidassociated virulence factors.Although pO157 has been reported in most O157 H7 strains (13), our study demonstrates a high variability in the putative virulence factor content of pO157 as well as a highly variable content of pO157-associated virulence factors among the O157 isolates screened.Finally, pO157-associated virulence factors were detected among all but 4 of the 20 E. coli serotypes examined.

Table .
Virulence factor targets and primers, including nucleotide sequences, reference, and PCR conditions*