STEC Isolation and Identification Protocol

The STEC strains isolation protocol used in this study was described previously [13 (link)]. In brief, the swab including rinsate was enriched with TSB at 25°C for 2h and 42°C for 8h. For O157 STEC, the enrichment was subjected to immunomagnetic separation (IMS), followed by plating on selective media, Sorbital MacConkey (Difco Labs: Detroit, MI) with cefixine (0.05μg/mL; Introgen/Dynal) and tellurite (2.5μg/mL; Introgen/Dynal) (CT-SMAC) and Rainbow Agar O157 (Biolog, Hayward, CA) containing novobiocin (20μg/mL; Sigma-Aldrich) and tellurite (0.8μg/mL; Introgen/Dynal) (NT-RA), for isolation. Subsequently, the presumptive colonies were screened for the presence of rfbE gene by polymerase chain reaction (PCR) assay. As for non-O157 STEC, similar methods were used, including IMS, for isolation. A parallel isolation method included PCR screening for stx genes and was conducted immediately after sample enrichment. Subsequently, the stx-positive enrichments were plated on CHROMagar O157 (DRG International, Mountainside, New Jersey). The presumptive colonies were then confirmed for the presence of stx genes by PCR. The serotypes of the isolated STEC strains were determined by enzyme-linked immunosorbent assay (ELISA), and confirmed by E. coli Reference Center at Penn State University.