Tryptone soy broth (tsb)

The strains of E. coli STCC 4201, Salmonella Typhimurium STCC 878, L. monocytogenes STCC 5672, and S. aureus STCC 4465 were obtained from the Spanish Type Culture Collection (STCC). The bacterial cultures were maintained frozen at –80°C in cryovials. A broth subculture was prepared by inoculating 10 mL of tryptone soy broth (Biolife, Milan, Italy) supplemented with 0.6% (w/v) yeast extract (Biolife) (TSBYE) with a loopful of growth from tryptone soy agar (Biolife) supplemented with 0.6% (w/v) yeast extract (TSAYE). The subculture was incubated at 35°C for 6–12 h in a shaking incubator (150 rpm; Heidolph Instruments, Vibramax 100, Schwabach, Germany). With these subcultures, 250 mL flasks containing 50 mL of TSBYE were inoculated to reach a concentration of 10⁴ CFU/mL and then incubated for 24 h under the same conditions until the stationary growth phase was reached (2 × 10⁹ CFU/mL).

Well-separated presumptive E. coli colonies were streaked on agar for isolation and cultured overnight in Tryptone Soy Broth (Biolife) with 0.3% Yeast Extract (Biolife). Lysates of overnight cultures were used as a DNA template in all PCRs protocols in this study. Identification of presumptive E. coli colonies (n = 378) was performed by amplifying the uidA gene62 (link). PCR products were separated by agarose gel electrophoresis (1%) and visualized with GelRed (Biotium).