Hybridizations were carried out at 65°C. To determine

the genetic relationship between the IncA/C plasmids, Pst I AZD5582 solubility dmso restriction profiles were analyzed with GelComparII. Clustering was performed using the UPGMA algorithm based on Dice coefficients. One reference isolate was run on all gels. A stringency parameter of 1.0% band position tolerance was used since this was the point at which the common restriction profile was identical across gels. PCR assays and nucleotide sequencing The complete list of primers used in this study is shown in Additional file 1, Table S1. To determine the incompatibility learn more groups of the plasmids, PCR-replicon typing for the Salmonella isolates and their E. coli transformants was performed using the primers and conditions recommended by Carattoli et al. [21]. The incompatibility groups tested were IncA/C, FII, HI1, HI2 and I1. The E. coli transformants carrying the IncA/C plasmids were screened by PCR using Crenigacestat purchase primers to detect seven regions

distributed throughout the reported IncA/C plasmids [5–8, 10] (Figure 3). The primers used are listed in Additional file 1, Table S1, and for a detailed explanation see the legend to Figure 3. The nucleotide sequences of these regions were determined for a representative sample of ten isolates (Additional file 2, Table S2) using the same primers and conditions. Plasmid DNA of the transformants was used for PCR mapping of the

CMY island and surrounding regions. Overlapping PCR assays were designed to cover the CMY region using primers previously published [33] or designed by us based on the reported sequence of pSN254 Leukocyte receptor tyrosine kinase [GenBank:NC_009140] [8]. Nine reactions were designed to determine the configuration at the CMY region (Figure 4, PCRs A-I). PCRs A, B, D and G were included in the plasmid PCR screening scheme to examine the CMY junction of all isolates. The nucleotide sequence for the 12,563 bp CMY region was generated for isolate YUHS 07-18 [GenBank:HQ203988], which was the most recent representative isolate of ST213. Accession numbers of the nucleotide sequences generated for representative strains (Additional file 2, Table S2) are as follows: repA/C [GenBank: HQ203980], floR [GenBank: HQ203981], PCR G [GenBank: HQ203982], PCR A [GenBank: HQ203983], R-7 [GenBank: HQ203984], R-8 [GenBank: HQ203985], and two mer alleles [GenBank: HQ203986] and [GenBank: HQ203987]. All nucleotide sequences were compared against public databases using the BLAST algorithm at NCBI [34]. Conjugation experiments We performed conjugation experiments for 17 Typhimurium isolates using a rifampicin (100 μg/ml)-resistant derivative of E. coli DH5α as the recipient.