Antimicrobial susceptibility testing Susceptibility to a standard panel of antimicrobial agents http://www.danmap.org was determined by microbroth dilution and interpreted according to Clinical and Laboratory Standards Institute guidelines [32], except for ciprofloxacin (≥0.125 μg/mL was used as breakpoint). Phage typing Phage typing was performed by the National Food Institute, Technical University of Denmark according to the phage typing scheme developed by Callow [33] and extended by Anderson et al. [34]. Strains that react with phages, but have a profile not included in the phage learn more typing scheme, are denoted Reaction Does Not Conform (RDNC). DNA microarray The DNA microarray used
in this study was previously find more described [35]. A set of 281 gene-specific
57-60 mer oligonucleotide probes were designed using the program Array Designer 4.1 (Premier Biosoft, Palo Alto, CA, USA). The oligonucleotides were spotted on glass slides using a QArray Mini Arrayer (Genetix, New Milton, UK). Hybridized spots were visualized in a GenePix Selleck CB-839 4000B laser scanner (Axon, Foster City, CA). Each oligonucleotide allows the detection of the presence or absence of a characteristic sequence previously described in Salmonella. The microarray used gives no information on the location of a gene or target sequence and can only score its presence or absence. Uncertain array results were resolved by PCR using primers described previously [35]. Data analysis Analysis of the DNA microarray data was performed as previously described [35]. A comparison was made by importing array values, gene present or absent, into BioNumerics 5.1 (Applied Maths, Sint-Martens-Latem, Belgium) as character data. An Unweighted Pair Group Method with Arithmetic mean (UPGMA) dendrogram (Fig. 2) was calculated by simple matching of binary coefficients on the basis of a geneset consisting of all genes from the array except the serotyping markers and the resistance markers. Multiple-locus
variable-number of tandem-repeat analysis (MLVA) MLVA was performed as described previously [36] to ensure that the strains were likely to be epidemiologically unrelated. The MLVA repeats were calculated over and named according to the method described recently [37]. Pulsed-field gel electrophoresis (PFGE) PFGE was carried out with XbaI restriction enzyme according to the Pulse-Net protocol [38], gels were analyzed in BioNumerics 5.1, and profiles were assigned by comparison to a database with known Danish profiles (see additional file 1: Xba I PFGE profiles of all isolates). Sequence typing Multilocus sequence typing (MLST) was carried out as previously described [39] and the alleles and the sequence types were assigned according to the MLST scheme on http://mlst.ucc.ie/mlst/mlst/dbs/Senterica/. Acknowledgements EL was partly funded by the Danish Research Council. We are grateful to the patients without whose kind participation this study would not have been possible.