C. jejuni and C. coli species identification was confirmed using multiplex PCR as described previously [55]. Testing for susceptibility against tetracycline, streptomycin, kanamycin and nalidixic acid was conducted using the agar dilution method [52, 53]. The test ranges used were 0.06-32 μg/ml for tetracycline (Sigma), 0.125-64 μg/ml for Blebbistatin ic50 streptomycin (Sigma) and kanamycin (Amresco, Solon, Ohio), and 0.25-128 μg/ml for nalidixic acid (Sigma). The quality

control strain used was C. jejuni ATCC #33560 [11, 53]. For streptomycin and kanamycin testing, Escherichia coli ATCC #25922 and C. jejuni ATCC #33560 were included. Campylobacter isolates were defined as resistant or sensitive based on breakpoints of ≥ 16 μg/ml for tetracycline, ≥ 64 μg/ml for nalidixic acid, and ≥ 64 μg/ml for streptomycin and kanamycin [54, 56]. Fla typing Fla typing (n = 100) was carried out using the method of Nachamkin et al. [57] with ABT-888 concentration minor modifications. Whole cell lysate [58] was used as the template. PCR amplification was performed in a Mastercycler gradient 5331 thermocycler (Eppendorf, Hamburg, Germany). C. jejuni ATCC #700819 was used as the find more positive control, and sterile water was substituted for the DNA template as the negative control. To confirm the presence of the 1.7 kb flaA amplicon, 10 μl of the PCR product was subjected to gel

electrophoresis followed by ethidium bromide staining and UV transillumination. DdeI (Promega, Madison, Wis.) was used to digest 5 μl of the flaA PCR product according to the manufacturer’s instructions at 37°C for 12-16 h overnight. Digested samples were electrophoresed on a 2% agarose gel, followed by staining in 0.5 μg/ml ethidium bromide solution and UV transillumination. A 100 bp ladder (Promega) was used as a molecular size standard. Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis (PFGE) was performed using the PulseNet method [59] with slight modifications. Salmonella enterica serotype Braenderup H9812 (ATCC

#BAA-664) was used as the molecular weight size standard. Restriction Endonuclease digestion of each sample plug slice was carried out in a 100 μl mixture containing 85 μl sterile water, 10 μl 10× J buffer, 4 μl of 10 U/μl SmaI (Promega), and 1 μl BSA at 25°C for 3 h. Electrophoresis was performed using the Chef Mapper system (Bio-Rad, Hercules, Calif.) and the following conditions: auto algorithm function (50 kb low molecular weight and 400 kb high molecular weight), run time 18 h, initial switch time 6.76 s and final switch time 38.35 s. Gels were stained with 1 μg/ml ethidium bromide solution for 30 min, destained in 500 ml reagent grade water for 60-90 min with water changes every 20 min, and viewed under UV transillumination.