0). Data was analyzed with ABI Sequencing Analysis software (Version 5.1.1). The primers used for sequencing are listed in Table 6. In total, four PCR reactions and eight sequencing reactions were conducted for each isolate being typed. Additionally, one internal sequencing reaction was required for 14/26 S. Typhimurium CRISPR2 alleles, due to the increased length of this locus. There were two alleles (only representing 2/86 S. Typhimurium isolates), 181 and 205, which required
extra primers due to the presence of a Selleck LY2606368 duplicated region of the locus. The positions of these extra primers are shown in Additional file 1: Figure S1. CRISPR2 alleles that were sequenced using more than two primers are indicated in Table 3. Sequence analysis and sequence type assignment Sequences were assembled and aligned using SeqMan
and MegAlign, respectively (Lasergene 10, DNA Star, Madison, WI) and unique alleles were assigned a unique numerical designation. All sequences from this study were submitted as a batch to NCBI and the accession numbers (KF465853 – KF465929) are shown for each allele in Additional file CYT387 nmr 2. For each isolate the combination of allelic types at all four loci defines the serovar-designated sequence type (ST) (Tables 2 and 3), with each unique allelic type assigned a different ST number. The presence of a SNP in any marker was sufficient to INCB28060 ic50 define a new allele. Analysis of CRISPR1 and CRISPR2 was performed using CRISPR-finder (http://crispr.u-psud.fr/Server/). We did not identify any SNPs within either CRISPR locus that defined any allele. Allelic differences occurred from deletion of one or more spacers, addition of a spacer or duplication/triplication of a spacer. Discriminatory power was calculated using the method described by Hunter and Gaston , with strains defined as either unique STs or unique PFGE patterns. Relationships between TSTs were pheromone calculated using BURST (http://www.pubmlst.org/analysis/), with a group definition of n-1. Unique PFGE patterns, or pulsotypes, were defined by PulseNet, using the Dice
coefficient with an optimization of 1.5% and a position tolerance of 1.5%. The difference of one band is sufficient to call two PFGE patterns different. PFGE dendrograms were generated using BioNumerics v. 6.6. S. Typhimurium outbreak study A summary of 30 S. Typhimurium outbreak isolates that were obtained from the Pennsylvania Department of Health is listed in Table 4. Ten of these isolates associated with an outbreak in 2004 (cluster 0411PAJPX-1c) where affected patients had been on a bus trip together, though no vector was ever identified. Another 10 isolates were linked to an outbreak in 2009 (cluster 0905PAJPX-1), which was associated with live poultry. The remaining 10 isolates represent sporadic case isolates, also from 2009 but were not associated with the 0905PAJPX-1 outbreak and thus served as controls.