A Comparison of Culture- and PCR-Based Methods to Detect Six Major Non-O157 Serogroups of Shiga Toxin-Producing Escherichia coli in Cattle Feces

Abstract

Culture-based methods to detect the six major non-O157 (O26, O45, O103, O111, O121 and O145) Shiga toxin-producing E. coli (STEC) are not well established. Our objectives of this study were to develop a culture-based method to detect the six non-O157 serogroups in cattle feces and compare the detection with a PCR method. Fecal samples (n = 576) were collected in a feedlot from 24 pens during a 12-week period and enriched in E. coli broth at 40° C for 6 h. Enriched samples were subjected to immunomagnetic separation, spread-plated onto a selective chromogenic medium, and initially pooled colonies, and subsequently, single colonies were tested by a multiplex PCR targeting six serogroups and four virulence genes, stx1, stx2, eae, and ehxA (culture method). Fecal suspensions, before and after enrichment, were also tested by a multiplex PCR targeting six serogroups and four virulence genes (PCR method). There was no difference in the proportions of fecal samples that tested positive (74.3 vs. 77.4%) for one or more of the six serogroups by either culture or the PCR method. However, each method detected one or more of the six serogroups in samples that were negative by the other method. Both culture method and PCR indicated that O26, O45, and O103 were the dominant serogroups. Higher proportions (P < 0.05) of fecal samples were positive for O26 (44.4 vs. 22.7%) and O121 (22.9 vs. 2.3%) serogroups by PCR than by the culture method. None of the fecal samples contained more than four serogroups. Only a small proportion of the six serogroups (23/640; 3.6%) isolated carried Shiga toxin genes. The culture method and the PCR method detected all six serogroups in samples negative by the other method, highlighting the importance of subjecting fecal samples to both methods for accurate detection of the six non-O157 STEC in cattle feces.

Prevalence and Level of Enterohemorrhagic Escherichia coli in Culled Dairy Cows at Harvest

“…When a pool was positive for stx, the DNA preparation from each isolate in the pool was individually tested with an 11-plex PCR assay. This 11-plex PCR assay included genes representing each of the EHEC-7 serogroups (wzx, wbq, or rfbE) plus stx 1 , stx 2 , and EHEC-hemolysin (ehxA) (4), which was modified by the use of primers for eae as described by Blanco et al (9) and primers for the wzx gene of O111 as described by Noll et al (53). Additional aliquots of broth enrichment culture of each fecal and hide and carcass surface sample were obtained and held at 48C (24 to 96 h) until molecular screening assays (Atlas and NS) were completed ( Fig.…”

“…Up to 10 target colonies were picked per plate and heated at 958C in 50 ll of water for use as DNA template. Colonies were tested with the 11-plex PCR assay (4,9,53). Colonies positive for one of the EHEC-7 serogroup genes plus stx and eae were confirmed as an EHEC-7 strain, and the sample was counted as positive.…”

Comparison of Agar Media for Detection and Quantification of Shiga Toxin–Producing Escherichia coli in Cattle Feces

“…Fifty microliters of nondiluted or 10-fold diluted bead suspension was spread plated onto mPossé2 and 50 ll of 100-fold bead suspension was spread plated onto mSHIBAM and incubated at 378C for 18 h. On mPossé2, 20 red/blue-purple and green colonies were picked and 20 enterohemolytic colonies were picked from mSHIBAM. Colonies were tested by multiplex PCR as described above (2,5,26).…”

“…In total, 10 or fewer colonies per plate of the target phenotype were picked, and each colony was suspended in 50 ll of water. The suspended colonies were heated for 10 min at 958C and tested by multiplex PCR (2,5,26). Concentration was determined by the proportion of colonies that tested positive for Ogroup, stx, and eae that matched that of the inoculum.…”

“…and Noll et al(26), respectively. Each reaction contained 10 ll of iQ multiplex powermix (Bio-Rad, Hercules, CA), 1 ll of all primers mixed, resulting in final primer concentrations of 0.21 lM; 2 ll of water; and 7 ll of DNA template.…”