Preston broth

C. jejuni ATTCC 33291 was cultured in Preston broth (Oxoid, Hampshire, UK) under microaerobic conditions (10% CO₂, 5% O₂, and 85% N₂) at 42 °C for 24 h. The procedure used for the preparation of inoculum and inoculation of chicken legs were previously described [25 (link)].

In total, 118 duck samples (carcasses and meat) were collected from a duck slaughterhouse (n = 40) and retail markets (n = 78) from January 2017 to July 2018. Forty samples from the slaughterhouse were obtained by swabbing each duck carcass with sterilized gauzes soaked with saline. The swab samples were then applied to enrichment broth including Bolton Broth (Oxoid Ltd., England, UK) and Preston Broth (Oxoid Ltd., England, UK) and cultured at 42 °C for 24 h under microaerobic conditions (6% O₂, 7.1% CO₂, 3.6% H₂, and 83.3% N₂). For the 78 samples from retail markets, duck meat was submerged in enrichment broth and cultured at 42 °C for 24 h under microaerobic conditions. The enriched broth was streaked on modified charcoal cefoperazone deoxycholate (mCCD) agar (Oxoid Ltd., England, UK) and Preston agar and incubated at 42 °C for 24 h under microaerobic conditions. Next, Campylobacter-like colonies were picked, and template DNA was prepared. To identify C. coli, polymerase chain reaction (PCR) was performed using primers designed on Campylobacter 16s rDNA and ask genes (Table S1).

C. jejuni K49/4 was isolated from poultry meat, identified phenotypically, and subcultured prior to the experimental test conditions. Microaerobic growth (5% O₂, 10% CO₂, 85% N₂) in Preston broth (Oxoid, Hampshire, UK) containing 5% (v/v) defibrinated horse blood (Oxoid) at 42°C for 9 h induced the entry of these cultures into the exponential growth phase, and for 15 h, into the stationary growth phase (1 (link)). To produce heat-shock stress, the bacterial cells harvested from each growth phase were exposed to temperature shifts from 42°C to 48±1°C or 55±1°C using thermoblock (Eppendorf Thermomixer comfort, Eppendorf AG, Hamburg, Germany) for 3, 10, 20, or 30 min, and these cells were then shortly cooled on ice to achieve 42°C before being analyzed. Untreated C. jejuni from both growth-phase cultures were used as controls and kept at 42°C throughout the experiment. To evaluate resistance to the heat treatments, 5 μg mL⁻¹ chloramphenicol was added as an inhibitor of protein synthesis before 5 h of starvation and the subsequent heat stress. C. jejuni cells were harvested by centrifugation (12,000×g for 5 min at 4°C), washed, resuspended in Ringer’s solution supplemented with 5 mM KH₂PO₄ (Kemika, Zagreb, Croatia), and incubated microaerobically for 5 h at 42°C for pre-starvation.