The following O157 strains were used in peptide susceptibility assays: (i) EDL933 (ATCC 43895; stx1+, stx2+, eae+, hlyA+; American Type Culture Collection/ATCC, Manassas, VA) and (ii) 86–24 (NADC 6103; stx2+, eae+). O157 strains EDL933 and 86–24 displayed similar results in initial peptide susceptibility assays, and due to EDL933 having both stx genes, only strain EDL933 was used in subsequent assays. Bacterial freezer stocks were streaked individually on Luria-Bertani (LB) Lennox agar (Sigma-Aldrich, St. Louis, MO) and incubated overnight at 37˚C. Bacterial cultures were prepared by growing each isolate from a single colony in 5 mL LB broth (Sigma-Aldrich) per assay for 20 h at 37˚C with constant shaking at 150 rpm. Assay isolates were streaked on Difco sorbitol MacConkey agar (BD Biosciences, Franklin Lakes, NJ) containing 4-methylumbelliferyl-β-d-glucuronide (100 mg/L; Sigma-Aldrich) (SMAC-MUG) or SMAC-MUG supplemented with cefixime (50 μg/L), potassium tellurite (2.5 mg/L), and vancomycin (40 mg/L) (SMAC-CTMV) agar [32 (link), 33 (link)]. Plates were read after overnight incubation at 37˚C and colonies that did not ferment sorbitol or utilize 4-methylumbelliferyl-β-d-glucuronide (non-fluorescent under UV light) were confirmed to be O157 via latex agglutination (E. coli O157 latex, Oxoid Diagnostic Reagents, Oxoid Ltd., Hampshire, UK) [32 (link), 33 (link)].
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