01 (Applied Maths, Sint-Martens-Latem, Belgium). The consensus sequences were queried against the pubMLST database to determine the allele PD332991 designations and Sequence Type (ST) of each

isolate. Sequences of new alleles and new allelic profiles were submitted to the pubMLST database and were assigned new numerical identifiers. As observed by others, amplification and sequencing of gyrB and recA with the original primers has not always led to results [17]. Therefore, each of these genes was divided into two fragments (gyrB-up, gyrB-down, recA-up, and recA-down). Two inner primers were designed (gyrB-up_rev: [M13-rev]CGATTCAACCGCTGATTTCACTTC; Z-VAD-FMK molecular weight gyrB-down_for: [M13-for]GCGGCACTAACACGTACGCTAAAC; recA-up_rev: [M13-rev]ACGGATTTGGTTGATGAAGATACA; recA-down_for: [M13-rev]GGGTCTCCAAGCTCGTATGC) and ‘5′-tailed’ with the universal M13 primers (M13-for: TGTAAAACGACGGCCAGT APR-246 price and M13-rev: CAGGAAACAGCTATGACC).

This enabled PCR amplification and sequencing with the conditions and in combination with the original primers published by González-Escalona et al.[13]. Peptide sequence type designation Translating the in-frame nucleotide sequences into the peptide sequences allows an analysis on the phenotypic level, as only non-synonymous substitutions of nucleotides leading to a different amino acid were considered. Similar to the nucleotide sequences, each unique peptide sequence was assigned a distinct numerical identifier and the oxyclozanide different combinations of alleles at each locus lead to the allelic profile at peptide level. Each individual profile was transformed to a peptide Sequence Type (pST) that allows the unambiguous identification of a clone. The peptide sequences and peptide profiles of the entire pubMLST dataset were submitted to the pubMLST database and implemented as an additional typing scheme, called AA-MLST, accessible at the pubMLST web page [32]. The loci

were labeled with the prefix ‘p_’ and the appropriate locus designation. Data analysis Phylogenetic analysis The generated sequence data were analyzed using Bionumerics and compared to already accessible sequences on the pubMLST web page [32]. To visualize the clonal relationship between isolates of subsets and in context with the entire dataset stored in the pubMLST database the goeBURST algorithm was used [33, 34]. By using the allelic profile data – on nucleotide and peptide level, respectively – isolates were subdivided into groups of related genotypes. Isolates that shared 100% identity in 6 of the 7 loci with at least one other member of the group, the single locus variants (SLVs), were assigned to a single clonal complex (CC). The algorithm also predicted the presumable founder (p)ST of each CC and any single and double locus variants originating. The algorithm was also used to obtain a ‘population snapshot’ with the group definition 0 of 7 loci shared and to create a fullMST, where all STs were connected [34, 35].