Collected samples were transported and/or stored 12 to 24 hr on ice until the isolation method was initiated, usually the morning after collection. Our method was modified from a method described previously for isolation of EcO157 from cattle feces, carcass and hide samples [44] (link). Ten grams of sample, including soil, sediment, feces or tissue (plant or colon), were removed by hand using clean exam gloves or with a sterile spatula, and placed into a 250 ml sterile flask containing 90 ml tryptic soy broth (TSB; Becton Dickinson, Sparks, MD). Moore swabs were rinsed briefly in tap water to remove mud and sediment, and were added to sterile flasks (1 liter) containing 250 ml TSB for enrichment. The flasks were incubated for two hrs at 25°C with shaking at 200 RPM, then at 42°C for 8 hrs, and held at 4°C without shaking until the following morning. Water samples were enriched by adding 11 ml of 10× TSB (filter sterilized, 0.45 µm) to 100 ml of sample, and the mixture was incubated as described above.
One ml of the enrichment broth was tested by adding 20 µl of anti-O157 antibody bound to magnetic beads (Invitrogen/Dynal, Carlsbad, CA) and mixing them for 30 min. The beads were washed twice with PBS containing 0.05% Tween 20 (PBS-T), then they were re-suspended in 100 µl PBS [45] . Alternately, beads were incubated and wash using the Dynal BeadRetriever (Invitrogen/Dynal, Carlsbad, CA) using the EPEC/VTEC Protocol. With either method, 50 µl each of the resuspended beads were spread on Sorbitol MacConkey agar (Difco Labs; Detroit, MI) containing cefixime (0.05 µg/ml; Invitrogen/Dynal) and tellurite (2.5 µg/ml; Invitrogen/Dynal) (CT-SMAC), and on Rainbow Agar (Biolog, Hayward, CA) containing novobiocin (20 µg/ml; Sigma-Aldrich) and tellurite (0.8 µg/ml; Invitrogen/Dynal) (NT-Rainbow). To aid in identification of authentic EcO157 colonies, plates of both media were streaked with an EcO157 strain RM2315 marked with the Green Fluorescent Protein [46] (link). Plates were incubated at 37°C overnight (approximately 18 hr).
Suspect colonies and positive control EcO157 strain RM2315, were patched onto duplicate plates of Luria Broth (LB) agar (Fisher Scientific, PA) using a numbered grid and incubated at 37°C overnight. One set of the patched colonies was blotted onto Protran nitrocellulose membranes (BA 85, Whatman/Schleicher & Schuell; Sanford, ME). The membrane was washed with 25 mM TRIS pH 7.4, 0.15 M NaCl, 0.1% Tween 20 (TBS-Tween) and blocked by immersion in 10 ml of 0.5% casein, 0.01 M TRIS, 0.031 M sodium azide, 0.15 M NaCl pH 7.4 (blocker) with shaking for 30 minutes at RT. The membrane was incubated in anti-O157 IgG monoclonal antibody (MAb), 13B3 [47] (link), diluted 1∶2000 in 10 ml 1× TBS-Tween containing 1% BSA, 0.02% KCl, 0.1% sodium azide (ELISA Diluting Buffer) and with shaking for 30 minutes at RT. The membrane was washed four times with 100 ml TBS-Tween, then incubated in goat anti-mouse IgG conjugated with alkaline phosphatase (Invitrogen/Zymed, Carlsbad, CA) diluted 1∶2000 in ELISA Diluting Buffer for 30 minutes with shaking at RT. The membrane was washed four times with TBS-Tween, then twice in distilled water. The membrane was developed by the addition of 10 ml of a 0.15 mg/ml and 0.3 mg/ml solution of 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) and Nitro Blue Tetrazolium (NBT) Sigma-Aldrich, B5655), respectively. During multiple isolation procedures, we noted that authentic EcO157 colonies were distinguishable from apparent antibody-positive non-EcO157 bacteria by the rate of the color reaction compared to EcO157 strain RM2315.
One ml of the enrichment broth was tested by adding 20 µl of anti-O157 antibody bound to magnetic beads (Invitrogen/Dynal, Carlsbad, CA) and mixing them for 30 min. The beads were washed twice with PBS containing 0.05% Tween 20 (PBS-T), then they were re-suspended in 100 µl PBS [45] . Alternately, beads were incubated and wash using the Dynal BeadRetriever (Invitrogen/Dynal, Carlsbad, CA) using the EPEC/VTEC Protocol. With either method, 50 µl each of the resuspended beads were spread on Sorbitol MacConkey agar (Difco Labs; Detroit, MI) containing cefixime (0.05 µg/ml; Invitrogen/Dynal) and tellurite (2.5 µg/ml; Invitrogen/Dynal) (CT-SMAC), and on Rainbow Agar (Biolog, Hayward, CA) containing novobiocin (20 µg/ml; Sigma-Aldrich) and tellurite (0.8 µg/ml; Invitrogen/Dynal) (NT-Rainbow). To aid in identification of authentic EcO157 colonies, plates of both media were streaked with an EcO157 strain RM2315 marked with the Green Fluorescent Protein [46] (link). Plates were incubated at 37°C overnight (approximately 18 hr).
Suspect colonies and positive control EcO157 strain RM2315, were patched onto duplicate plates of Luria Broth (LB) agar (Fisher Scientific, PA) using a numbered grid and incubated at 37°C overnight. One set of the patched colonies was blotted onto Protran nitrocellulose membranes (BA 85, Whatman/Schleicher & Schuell; Sanford, ME). The membrane was washed with 25 mM TRIS pH 7.4, 0.15 M NaCl, 0.1% Tween 20 (TBS-Tween) and blocked by immersion in 10 ml of 0.5% casein, 0.01 M TRIS, 0.031 M sodium azide, 0.15 M NaCl pH 7.4 (blocker) with shaking for 30 minutes at RT. The membrane was incubated in anti-O157 IgG monoclonal antibody (MAb), 13B3 [47] (link), diluted 1∶2000 in 10 ml 1× TBS-Tween containing 1% BSA, 0.02% KCl, 0.1% sodium azide (ELISA Diluting Buffer) and with shaking for 30 minutes at RT. The membrane was washed four times with 100 ml TBS-Tween, then incubated in goat anti-mouse IgG conjugated with alkaline phosphatase (Invitrogen/Zymed, Carlsbad, CA) diluted 1∶2000 in ELISA Diluting Buffer for 30 minutes with shaking at RT. The membrane was washed four times with TBS-Tween, then twice in distilled water. The membrane was developed by the addition of 10 ml of a 0.15 mg/ml and 0.3 mg/ml solution of 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) and Nitro Blue Tetrazolium (NBT) Sigma-Aldrich, B5655), respectively. During multiple isolation procedures, we noted that authentic EcO157 colonies were distinguishable from apparent antibody-positive non-EcO157 bacteria by the rate of the color reaction compared to EcO157 strain RM2315.
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