Moreover, to expand our knowledge on virulence plasmid diversity, in this study, we also report the nucleotide sequence of the second most frequently isolated vapA-carrying plasmid type: an 87-kb type I plasmid. In this study, we used a total of 96 R. equi field strains, comprising 61 clinical strains isolated from horses that had been autopsied in Normandy (France) from 1995 to 2006 and whose death had been caused by R. equi, 22 strains isolated from organic samples collected at horse-breeding farms (faeces, straw and manure) and 13

environmental strains isolated from horse-breeding farm environments Copanlisib cost (water, soil and dust). For more details, see Supporting Information, Table S1. Field strains were identified as R. equi based on colony morphology, API Coryne (bioMérieux, France) biochemical profiling and a synergistic haemolysis (CAMP-like) test with Listeria ivanovii (Navas et al., 2001). The bacteria were grown for 24 h at 37 °C using brain–heart infusion (BHI) broth (BD-Difco) as the base medium, supplemented with 1.5% agar for plate cultures. Plasmid DNA was isolated from R. equi using the alkaline lysis method (Sambrook et al., 1989) with the following modifications: R. equi strains were grown for 24 h at 37 °C in 20 mL of BHI broth (BD-Difco). Ten millilitres of bacterial cultures were centrifuged, and the bacterial pellet was washed in 5 mL of 0.5 M Tris-HCl (pH

Androgen Receptor Antagonist manufacturer 8.0). After centrifugation, pellets were resuspended in 200 μL of a freshly prepared solution containing 0.025 M Tris-HCl (pH 8.0), 0.01 M EDTA (pH 8.0) and 0.05 M glucose, plus 20 mg mL−1 lysozyme.

The bacteria were incubated at 37 °C for 1 h. Cells were then lysed by adding 400 μL of a solution containing 1.0% w/v sodium dodecyl sulphate and 0.2 M NaOH. Chromosomal DNA was precipitated Megestrol Acetate with 5 M potassium acetate acetic acid buffer (pH 4.8) and centrifuged at 13 000 g for 5 min. Then, supernatants were transferred to MaXtract high-density gel barrier tubes (Qiagen), and an additional phenol–chloroform extraction, followed by chloroform extraction, was carried out according to the manufacturer’s instructions. Plasmid DNA was digested with BamHI, EcoRI and HindIII endonucleases to generate RFLP profiles. DNA fragments were separated by electrophoresis in 0.7% agarose gels at approximately 5 V cm−1 for 2 h, and visualized using ethidium bromide under UV transilluminator. All chemicals were purchased from Sigma-Aldrich unless stated otherwise. Plasmid sequencing was performed by Qiagen Sequencing Services, Germany (>99.99% accuracy). The sequence of pVAPA116 was submitted to the GenBank™/EMBL data base (accession number HM114217). The sequence of pVAPA116 was compared with that of other plasmids using tblastx (Abbott et al., 2005) and comparisons were visualized with the Artemis comparison tool (ACT) (Carver et al., 2005). Identification of horizontally acquired DNA was performed using the Alien Hunter algorithm(http://www.sanger.ac.