The amplification reactions were performed in 20 μl using 2 μl DNA extract (approximately 20 ng

of DNA) as a template. Real-time PCR reactions were performed in a LightCycler® 480 System using LightCycler® 480 SYBR Green I Master (Roche Diagnostics GmbH, Germany) according to recommendations given by the manufacturer of find more the kit. The temperature program was as follows: 5 min initial denaturation at 95°C followed by 35 cycles of denaturation at 95°C for 10 s, annealing at 56°C for 10 s and primer extension at 72°C for 30 s. The amplifications were terminated after a final elongation step of 7 min at 72°C. The PCR fragments were verified by electrophoresis using Bioanalyzer (CHIR-99021 mw Agilent Technologies, USA). PCR products were purified and sequenced by Eurofins MWG Operon

(Ebersberg, Germany) using the dideoxy chain termination method on a ABI 3730XL sequencing instrument (Applied Biosystems, STI571 USA). Data analysis The Staden Package [44] was used for alignment, editation and construction of consensus sequences based on the ABI sequence chromatograms. Consensus sequences were entered into the MEGA4 [45] software and aligned by CLUSTALW [46]. Sequences were trimmed to be in frame and encode an exact number of amino acids. Dendograms for each locus (Additional triclocarban file 1) were constructed in MEGA4 using

the Neighbor-Joining method (NJ) with branch lengths estimated by the Maximum Composite Likelihood method [45, 47]. Branch quality was assessed by the bootstrap test using 500 replicates. A subset of six loci including adk, ccpA, recF, sucC, rpoB and spo0A, which gave the highest tree resolution and still being congruent (visual evaluation, Additional file 1), was selected for the final MLST scheme (highlighted in Table  1). The trimmed sequences were entered into BioNumerics software v. 6.6, (Applied Maths NV) as fasta files and used to generate allelic profiles for each isolate based on the six loci. Each unique allelic profile defined a sequence type (ST). A cluster analysis was performed using the allelic profiles as categorical coefficients and a dendogram was constructed based on the UPGMA method.