*2.2. Campylobacter Isolation, Enumeration, and Identification*

Each poultry sample was placed into a sample bag containing 400 mL 0.1% buffered peptone water (BPW, Becton, Dickinson and Company, Sparks, MD, USA) and gently shaken for 60 s. For *Campylobacter* isolation, the rinsate (27 mL) was mixed with 27 mL 2 × blood-free Bolton broth (Oxoid Ltd., Basingstoke, Hampshire, UK) and the mixture was enriched at 42 ◦C for 48 h. Loopful portions (10 μL) of the enrichments were streaked on modified charcoal-cefoperazone-deoxycholate agar (mCCDA; Oxoid Ltd., Basingstoke, UK) and incubated at 42 ◦C for 48 h in a microaerobic environment (5% O2, 10% CO2, and 85% N2) created by CampyGenTM gas packs (Oxoid Ltd., Basingstoke, UK). The two presumptive *Campylobacter* colonies (gray, mucoid, and flat) on a plate were selected and each colony of them was streaked on two Colombia agar plates (bioMérieux, Marcy-l'Étoile, France) for aerobic and microaerobic conditions at 42 ◦C for 48 h under both aerobic and microaerobic conditions. The colonies grown under microaerobic conditions were further analyzed to identify *Campylobacter* by PCR using the primers listed in Table 1. To extract *Campylobacter* DNA, the presumptive colonies at plate were suspended in 0.2 mL of sterilized distilled water, and heated at 99 ◦C for 10 min. The suspensions were centrifuged at 14,000 rpm for 3 min, and supernatants were then used for PCR amplification. The program was as follows: pre-denaturation at 95 ◦C for 15 min, 25 cycles of denaturation at 95 ◦C for 0.5 min, annealing at 58 ◦C for 1.5 min, and extension at 72 ◦C for 1 min. A final extension step at 72 ◦C for 7 min was performed [26]. The PCR products were visualized by electrophoresis and UV-transillumination. The isolates were used in further experiments for analysis of antibiotic resistance, genetic diversity and *cdt* genes. To enumerate *Campylobacter* cells, 1 mL of the rinsate was serially diluted using 0.1% BPW, and 0.1 mL of aliquots were plated on mCCDA (Oxoid Ltd., Basingstoke, UK). The plates were then incubated at 42 ◦C for 48 h under microaerobic conditions. Five presumptive colonies on each plate were then analyzed by PCR using the conditions described above. The contamination levels of *Campylobacter* were determined by multiplying the number of positive colonies per five presumptive colonies to the total number of colonies. Additionally, each carcass was weighted to calculate the colony forming units per g (CFU/g).


**Table 1.** Primer sequences used to identify the *Campylobacter* genus and species.

## *2.3. Antibiotic Susceptibility Testing*

The isolated colonies were further analyzed for antibiotic susceptibility to chloramphenicol, amikacin, erythromycin, tetracycline, ciprofloxacin, nalidixic acid, and enrofloxacin (Sigma-Aldrich, St Louis, MO, USA), according to the guidelines of the Clinical & Laboratory Standards Institute [29]. To determine antibiotic resistance, the breakpoints suggested by CLSI [29], CDC [30], Hong et al. [31], and Kang et al. [32] were used as follows: chloramphenicol at 32 μg/mL, amikacin at 64 μg/mL, erythromycin at 32 μg/mL, tetracycline at 16 μg/mL, ciprofloxacin at 4 μg/mL, nalidixic acid at 64 μg/mL, and enrofloxacin at 4 μg/mL. The *Campylobacter* isolates on Colombia agar (bioMérieux, Marcy-l'Étoile, France) were suspended in Mueller-Hinton broth (MHB; Becton, Dickinson and Company, Sparks, MD, USA) to obtain a McFarland 0.5 standard, and further diluted 10-fold. Using needles, *Campylobacter* isolates were spotted on Mueller-Hinton agar (MHA; Becton, Dickinson and Company, Sparks, MD, USA) with 5% lysed horse blood plates (Oxoid Ltd., Basingstoke, UK), formulated at 0.5–128 μg/mL with seven antibiotics. The plates were incubated under microaerobic

conditions at 37 ◦C for 48 h. MIC was determined by colony formation on the plates and the reference strain used was *Campylobacter jejuni* ATCC33560.
