Sulfide indole motility medium

Isolation of Salmonella spp. was firstly performed by pre-enrichment of samples in lactose broth (Merck, Darmstadt, Germany) at 37 °C for 24 h. For selective enrichment, pre-enriched cultures were transferred into Selenite Cystine (SC) broth (Merck, Darmstadt, Germany) and Tetrathionate Brilliant Green bile (TBG) broth (Merck, Darmstadt, Germany), and incubated at 35 °C for 24 h. Then, these cultures were streaked onto Bismuth Sulphite agar (BSA) (Oxoid, Basingstoke, UK), Xylose Lysine Deoxycholate Agar (XLD) (Oxoid, Basingstoke, UK), and Hektoen Enteric agar (HEA) (Oxoid, Basingstoke, UK) as selective media and incubated at 35 °C for 48 h. Typical colonies were cultured on the slants of Tryptic soy agar (TSA) (Merck, Darmstadt, Germany) and subjected to biochemical tests using Lysine Iron agar (LIA) (Merck, Darmstadt, Germany), Triple Sugar Iron (TSI) agar (Merck, Darmstadt, Germany), Sulfide-Indole-Motility (SIM) medium (Merck, Darmstadt, Germany), and Christensen’s Urea agar (Merck, Darmstadt, Germany) (Table S1) [89 ].

Approximately 10 g of each fecal sample was diluted in 90 mL of BPW (Oxoid) and mixed for 30 s. A 15 mL aliquot of each fecal mixture was stored for later use. Next, 1 mL of enriched fecal sample was mixed with 20 μL of magnetic beads coated with an anti-O157 antibody and IMS was performed according to the manufacturer’s instructions (Oxoid). The bead suspension (100 μL) was then plated onto CT-SMAC agar (Oxoid). The plates were incubated at 37 °C for 24 h and presumptive E. coli O157 colonies were identified as E. coli O157 that did not ferment sorbitol. These colonies were picked and inoculated into nutrient agar slants at 37 °C for 24 h and stored in a refrigerator for further biochemical analysis. These isolates were further verified using conventional biochemical tests as described by Harrigan [62 ]. Tests were performed to examine indole production and motility using Sulfide Indole Motility (SIM) medium (Merck); citrate utilization with Simmons citrate agar (Merck); and methyl red and vogues-Proskauer using MR-VP medium (Merck) [63 ].