Columbia agar base

50 C. coli isolates of different origin were obtained from the Federal Institute for Risk Assessment (BfR) in Berlin, Germany. The bacterial isolates were cultured on Columbia agar base (Merck) supplemented with 5 % sheep blood (BA) and incubated at 42 °C under microaerophilic conditions (5 % O₂, 10 % CO₂, 85 % N₂) for 18 hours prior to genomic DNA extraction. Genomic DNA of all C. coli isolates was extracted using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions.
Species confirmation was performed using MALDI Biotyper system (Bruker Daltonics, Bremen, Germany). Results with MALDI Biotyper identification score values ≥2.000 were considered correct. Additionally multiplex PCR was used to discriminate between C. jejuni and C. coli [63 (link), 64 (link)].
The MLS-type was established using amplification and sequencing primers reported before [65 (link)]. The cycling conditions were 94 °C for 1 min, followed by 35 cycles of 94 °C for 120 s, 50 °C for 60 s, 72 °C for 60 s, followed by a final elongation step of 72 °C for 5 min [65 (link)]. Amplicons of the seven genes included in the C. jejuni/C. coli MLST scheme were sent for sequencing to Seqlab Sequence Laboratories GmbH (Göttingen, Germany) using 10 pmol of the respective sequencing primer.

C. jejuni ssp. jejuni isolates were stored as cryobank stocks (Mast Diagnostica, Reinfeld, Germany) at −80 °C. For usage in this study, they were cultured as one batch on Columbia agar base (Merck, Darmstadt, Germany) supplemented with 5% sheep blood (Oxoid Deutschland GmbH, Wesel, Germany) and incubated overnight at 42 °C under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂). Experiments were performed under biosafety level 2 conditions.

C. coli and C. jejuni strains were stored for long-term storage in Cryobank tubes at −80 °C (Mast Diagnostica, Reinfeld, Germany). For the experiments, they were incubated as one batch overnight under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) on Columbia agar base (Merck, Darmstadt, Germany) supplemented with 5% sheep blood (Oxoid Deutschland GmbH, Wesel, Germany). Experiments were carried out under biosafety level 2 conditions.