A total of 51 Salmonella isolates were recovered from 105 samples (15 from the first and 15 from the last chilling waters, and 75 from carcasses) collected at two out of nine duck slaughterhouses as well as 13 out of 41 chicken slaughterhouses located in different regions of South Korea. The first chilling water, the last chilling water, and five carcasses from each slaughterhouse were sampled. Bacteria were isolated from the samples according to the standard International Standardization Organization (ISO)-6579 method . Serotyping was performed by slide and tube agglutination using O and H antiserum (Difco, USA) according to the Kauffmann and White scheme . If two colonies showed the same serotypes and antimicrobial resistant patterns, only one colony was randomly chosen for analysis in this study.
Antimicrobial resistance of all 51 Salmonella isolates were evaluated using a disc diffusion test with the following discs (Difco): amikacin (An, 30 μg), ampicillin (Amp, 10 μg), chloramphenicol (C, 30 μg), ceftazidime (Caz, 30 μg), cephalothin (Cf, 30 μg), ciprofloxacin (Cip, 5 μg), cefotaxime (Ctx, 30 μg), cefazolin (Cz, 30 μg), cefepime (Fep, 30 μg), cefoxitin (Fox, 30 μg), gentamicin (Gm, 10 μg), imipenem (Imp, 10 μg), kanamycin (K, 30 μg), nalidixic acid (Na, 30 μg), norfloxacin (Nor, 10 μg), streptomycin (S, 10 μg), trimethoprim/sulfamethoxazole (Sxt, 1.25/23.75 μg), and tetracycline (Te, 30 μg). The results were evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . Escherichia coli ATCC 25922 was used as the control strain.
All 51 Salmonella isolates from the present study were further tested for amino acid changes in the QRDR, and screened for the presence of the gyrA and parC genes. Minimal inhibition concentrations (MICs) were determined for two antimicrobials, Na and Cip, belonging to the quinolone and fluoroquinolone classes of antimicrobials, respectively, using an agar dilution method according to CLSI guidelines .
DNA was isolated for further molecular studies using a generation capture column kit (Qiagen, Germany) and stored at -70°C before use. Fragments of the gyrA (F, 5′-TGTCCGAGATGGCCTGAAGC-3′; R, 5′-TACCGTCATAGTTATCCACG-3′) and parC (F, 5′-CTATGCGATGTCAGAGCTGG-3′; R, 5′-TAACAGCAGCTCGGCGTATT-3′) genes containing the QRDR associated with quinolone resistance were amplified by PCR and sequenced as previously described [5, 6]. The PCR products were purified and sequenced by Macrogen Inc. (Daejeon, South Korea). The nucleotide sequences were analyzed with the Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Information (NCBI) website (http://blast.ncbi.nlm.nih.gov/Blast.cgi). DNA sequences of the gyrA and parC genes were compared to those of native gyrA DNA (GenBank accession number X78977) and native parC DNA (GenBank accession number M68936), respectively.