Collection of samples
In July 2021, a total of 84 random samples of unpasteurized cow’s milk were purchased from retail markets located in three areas of Qazvin Province, Iran [10]. All samples were collected under strict sanitary conditions, and placed in an insulated icebox at 4 °C until delivery to the laboratory of food microbiology at Qazvin University of Medical Science for further analysis.
Isolation of Campylobacter spp.
Twenty-five milliliters of each milk sample was centrifuged at 20,000 × g for 35 min at 8 °C. After discarding the supernatant, the pellet was mixed with 45 mL of Bolton broth containing Campylobacter-selective supplement (HiMedia, India) and 5% defibrinated sheep blood (Baharafshan, Iran), and then incubated for 48 h at 42 °C under microaerophilic conditions (10% CO2, 85% N2 and 5% O2) using a Gas Pack C (Merck, Germany). Following incubation, a 30 µL aliquot of enriched cultures was streaked onto mCCDA (QUELAB,Canada) with antibiotic Campylobacter-selective supplement (Oxoid, UK) and incubated as previously mentioned. The suspected Campylobacter colonies were subjected to examination of morphology, oxidase, and catalase activity [11, 12]. Subsequently, these colonies were preserved in BHI broth with glycerol, and kept at − 80 °C for further investigation.
Identification of Campylobacter spp.
A boiling procedure was employed for the extraction of DNA from isolates. A multiplex PCR technique targeting the 16 S rRNA gene was employed for molecular identification. Specifically, the primer pair 0301 (F, CTT AAA GCN ATG ATA GTR GAY AAR) and 0304 (R, ACA GGR ATT CCR CGY TTT GTY TC), was used to target all Campylobacter species, and the isolates were differentiated as C.jejuni and C.coli using specific IpxA genes [13]. The PCR mixture reaction (20 µL) consisted of 10 µL Master Mix (Ampliqon, Denmark), 2 µL of each primer, 5 µL DNA template, and 1 µL deionized water. The PCR proceeded as follows: initial denaturation at 95 °C for 5 min, followed by 45 cycles of denaturation at 95 °C for 40 s, annealing at 55 °C for 40 s, and extension at 72 °C for 1 min. The final extension phase lasted 7 min at 72 °C. Subsequently, the PCR products underwent electrophoresis on a 1.5% w/v agarose gel in 0/5× TBE buffer with DNA-safe stain (CinnaGen, Iran), running at 110 V for 75 min. The results were photographed using a gel documentation system (NovinPars Co., Iran) [14]. C. jejuni ATCC 33,291 and C. coli ATCC 43,478 were used as control strains.
Genotyping by RAPD-PCR
In the RAPD genotyping analysis of Campylobacter isolates, a primer with the sequence 5′-CGCGTGCCAG-3′ was employed. A 25 µl reaction volume was prepared, consisting of 2 µl of DNA template (50 ng/µl), 12.5 µl of PCR master mix, 1 µl of primer (0.2 M/µl), and deionized sterile water was used for RAPD amplification. The thermal cycling program proceeded as follows: 95 °C for 5 min, 1 min at 36 °C, 4 min at 72 °C, and then 35 cycles of 95 °C for 1 min, 36 °C for 1 min, with a final extension at 72 °C for 4 min. Amplified RAPD-PCR products were electrophoresed on a 1.5% w/v agarose gel containing 0.5X TBE buffer with staining, running at 100 V for 1 h. The gels were visualized using a Gel Doc system. All analyses of UPGMA dendrograms were performed using PyElph and NTsys software [15].
Antibiotic susceptibility testing
In this study, eight antibiotic disks (Padtan teb) were used, including tetracycline (30 g), erythromycin (15 g), doxycycline (30 g), azithromycin (15 g), nalidixic acid (30 g), chloramphenicol (30 g), ceftriaxone (30 g), and amoxicillin/clavulanic acid (30 g) [16]. The disc diffusion technique was conducted using the Kirby-Bauer method on Mueller-Hinton agar, following CLSI guidelines [17]. CLSI Enterobacteriaceae breakpoints were used to interpret resistance.
Statistical analysis
We employed the Chi-squared test and Fisher’s exact test to assess significant differences (P value ≤ 0.05) among the incidence rates. We conducted these analyses using SPSS version 22.0.1 (SPSS, Chicago, IL, USA).
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