12�C16 ��g/mL), streptomycin (1�C16 ��g/mL), ciprofloxacin (0.06�C4 ��g/mL), tetracycline (0.25�C16 ��g/mL), erythromycin (0.5�C32 ��g/mL), nalidixic acid (2�C64 ��g/mL), and chloramphenicol (2�C32 ��g/mL). After inoculation, the plates were incubated at 42 ��C in microaerophilic atmosphere for 24 hours and then screened. C. jejuni strain NCTC 11351 was used as control.2.4. SequencingCampylobacter strains resistant to nalidixic acid and/or ciprofloxacin were sequenced to evaluate any Quinolone Resistance�CDetermining Region (QRDR) mutation of gyrA gene. The sequencing was performed as suggested by Zirnstein [14] using Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer instructions with the Thermal Cycler GenAmp 9700 (Applied Biosystems).

The product was purified by Agencourt CleanSEQ and Dye-Terminator Removal (Agencourt Bioscience Corporation, Madison, WI, USA). Sequencing was carried out with the Avant Genetic Analyzer 3100 (Applied Biosystems).2.5. Pulsed Field Gel Electrophoresis (PFGE)PFGE was performed according to the instructions of the 2009 U.S. PulseNet protocol for Campylobacter. Carfilzomib Bacteria, previously identified by PCR, were subcultured onto Columbia agar and embedded in agarose blocks (Seakem Gold agarose, Lonza, Rockland, ME, USA). The blocks were then lysed, washed, digested with SmaI restriction enzyme (Promega, Milan, Italy) and subjected to pulsed-field electrophoresis in 1% agarose gel (Seakem Gold agarose, Lonza) for 18 h (Chef Mapper II, Biorad Laboratories, Hercules, CA, USA).

Salmonella serovar Branderup H9812 was used as standard molecular weight size. After electrophoresis run, the gel was stained with Sybr Safe DNA gel stain (Invitrogen) and photographed at transilluminator (Alpha Innotech). The image analysis was performed using the program Bionumerics v. 6.6 (Applied Maths NV, Sint-Martens-Latem, Belgium). Pair comparisons and cluster analyses were carried out using the Dice correlation coefficient and the unweighted pair group mathematical average (UPGMA) clustering algorithm. The optimization tolerance was set at 2.5% and the position tolerance for band analysis was set at 1%.2.6. DNA MicroarrayBacterial DNA was labelled using the Bioprime DNA labelling system kit (Invitrogen Life Technologies, Milano, Italy) as described previously [15]. The labelling efficiency and the percentage of dye incorporation were quantified by scanning the DNA samples at wavelengths from 200 up to 700 nm using a NanoDrop Spectrophotometer (NanoDrop Products, ThermoScientific, Wilmington, DE, USA) and analyzing data with the internet�Cbased Percent Incorporation Calculator (http://www.pangloss.com/seidel/Protocols/percent_inc.html).