Bacterial strains and media - A total of twenty Campylobacter strains isolated from poultry origin (cloacal swabs and raw poultry meat) in Calcutta, India were used in this study. E. coli ATCC 25922 was used as a quality control organism in antimicrobial susceptibility testing. All Campylobacter strains and E. coli ATCC 25922 were grown on blood base agar no. 2 (Oxoid, Basingstoke, UK) supplemented with 5% (v/v) defibrinated horse blood (Nippon Bio-Supp. Center, Tokyo, Japan) under microaerobic conditions (5% O2, 10% CO2 and 85% N2) at 37oC for 48 hr. DNA preparation - Template DNA was prepared by the boiling method. Briefly, a loopful of bacteria collected from agar plate was suspended in 1 ml of TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 8.0)] followed by boiling of the suspension for 10 min, centrifugation at 12, 800 g for 5 min and 2 μl of supernatant was used as a PCR template. hipO gene PCR - The hippuricase gene (hipO) was amplified by PCR using primers. The PCR reaction contained appropriate concentration of primer sets, 0.2 mM each of dNTP mixture (dATP, dCTP, dGTP, and dTTP), 1X Ex Taq DNA polymerase buffer, and 1.0 U of Ex Taq DNA polymerase in 40 μl reaction volume. Amplification was performed on an Applied Biosystems GeneAmp PCR 9700 (Applied Biosystems). PCR products were analyzed by 1.5% agarose gel electrophoresis and bands were visualized with UV light after staining with ethidium bromide (1 μg/ml). Images were captured on a Bio-Rad Gel Doc system (Bio-Rad Laboratories, Hercules, CA, USA). 2.4. Sequencing of the 16S rRNA gene - PCR primers for amplifying 16S rRNA gene are described in Table 1. Reaction mixture was as described above. PCR product was purified by a QIAquick PCR Purification Kit according to the manufacturer’s instruction (QIAGEN GmbH, Hilden, Germany). The purified DNA was subjected to cycle sequencing reaction by using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with the primers used for 16S rRNA gene amplification and four additional primers, 16S520F (5'-GAGTTTGATCCTGGCTC-3'), 16S1100F (5'-GCAACGAGCGCAACCC-3'), 16S741R (5'-GTATCTAATCCTGTTTGC-3') and 16S1240R (5'-CCATTGTAGCACGTGT-3'), which can specifically bind inner region of 16S rRNA gene were also used. Nucleotide sequences were determined by using an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). The sequences obtained were analyzed using the DNA Lasergene software package (DNASTAR, Madison, WI, USA). Homology searches were performed against all sequences in the GenBank database by using the BLAST search engine, available through the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov). Multiplex PCR - The multiplex PCR assay for detection of cdtA, cdtB and cdtC genes of C. jejuni, C. coli and C. fetus was performed by gene specific primers (Asakura et al., 2008) are summarized in Table 1. All reactions contained appropriate concentrations of three primer sets, 0.2 mM each of dNTP mixture, 1X Ex Taq DNA polymerase buffer, and 1.0 U of Ex Taq DNA polymerase in a 40-μL reaction volume. PCR products were analyzed by 2% agarose gel electrophoresis and visualization of bands and capturing of image were done. 2.6. Antimicrobial agents and susceptibility testing. All Campylobacter strains were tested against ampicillin (10 μg), tetracycline (30 μg), azithromycin (15 μg), erythromycin (15 μg), chloramphenicol (30 μg), gentamicin (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), ofloxacin (5 μg), fosfomycin (50 μg) and sulphamethoxazole-trimethoprim (1.25 μg) by disk diffusion method as described by Luangtongkum et al. (2007) with some modifications. Again, all intermediately resistant and resistant Campylobacter strains confirmed by disk diffusion method except sulphamethoxazole-trimethoprim resistant strains were subjected to the determination of minimum inhibitory concentration (MIC) by agar dilution method with some modifications. The antimicrobial resistance break points (Minimum inhibitory concentration: MIC) used were those established by national antimicrobial resistance monitoring system (NARMS) in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines: ampicillin, ≥32 μg ml-1; tetracycline, ≥16 μg ml-1; azithromycin, ≥8 μg ml-1; erythromycin, ≥8 μg ml-1; chloramphenicol, ≥32 μg ml-1; gentamicin, ≥16 μg ml-1; nalidixic acid, ≥32 μg ml-1; ciprofloxacin, ≥4 μg ml-1; levofloxacin,≥8 μg ml-1; ofloxacin, ≥8 μg ml-1. Resistance to fosfomycin was considered when the MIC was ≥128 μg/ml . All susceptibility data were confirmed on at least two separate experiments. PCR amplification of the gyrA gene - PCR amplification of the gyrA gene was performed by PCR primers. Sequence determination - PCR products of the gyrA genes were purified by a QIAquick PCR Purification Kit according to the manufacturer’s instruction (QIAGEN GmbH, Hilden, Germany). The purified DNA was subjected to cycle sequencing reaction by using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and primers used for the gyrA genes amplification. The reactions were conducted in a GeneAmp 9700 thermal cycler (Applied Biosystems) in accordance with the manufacturer’s instruction. Nucleotide sequences were determined by using an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). Homology searches were performed against all sequences in the GenBank database by using the BLAST search engine, available through the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov). Nucleotide and amino acid sequence alignments were subsequently generated by MegAlign and ClustalW programs in the lasergene software package (DNASTAR, Madison, WI, USA). DNA Sequences of C. jejuni UA580 (GenBank accession number L04566) and C. coli RM 2228 (GenBank accession number NZ_AAFL01000007) were used for making comparison with the gyrA genes sequences obtained in this study.