Rifampicin-resistant APEC O78 Isolation and Culture

APEC O78 (GenBank accession no. CP004009) (Tim Johnson, University of Minnesota, Saint Paul, MN, USA) (57 (link)). Rifampicin resistant APEC O78 (Rif^R) was isolated on Luria-Bertani (LB) agar (Sigma-Aldrich, Inc. MO, USA)containing 50 μg/mL of Rifampicin (EMD Millipore, USA) (58 (link)). For the preparation of bacterial inoculums, APEC O78 (Rif^R) was grown overnight in LB media containing 50 μg/mL of Rifampicin at 37°C with shaking at 200 rpm. The bacteria were then diluted 1:100 in fresh LB broth and was incubated with shaking at 200 rpm at 37°C for 3 h. Logarithmic phase grown culture of Rif^R APEC O78 (OD₆₀₀ ~ 0.5) was washed twice with PBS and adjusted to the required concentration (OD₆₀₀ = 0.1).