5 μL of 10× buffer, 2 mM of MgCl2, 40 pmol of primer, 200 mM of each of four dNTPs, 200 ng of template genomic DNA and 1 U of Taq polymerase. The PCR reactions were carried out as follows: an initial denaturation at 94°C for 5 min followed by 40 cycles of denaturation

at 94°C for 1 min, annealing at 36°C for 1 min and extension at 72°C for 1 min 30 secs. Four different primers were used: M13, P3, P15 and OPA03U are listed in Table 2[36–38]. BOX-PCR typing was carried out with the BOX-A1R primer (Table 2) [39]. 200 ng of template genomic was mixed with 2 U of Taq polymerase, 200 mM of each of four dNTPs, 2.5 μl of dimethyl sulfoxide (DMSO), 0.8 μl of bovine serum albumin (10 mg ml-1) (Promega), 5 μl of 5× Gitschier buffer and 10 pmol of CBL0137 primer in a final volume of 25 μl.

After initial denaturation for 2 min at 95°C, 35 amplification cycles were completed, each consisting of 40 secs at 94°C, 1 min at 50°C, and 8 mins at 65°C. A final extension of 8 mins at 65°C was applied. Amplified products for both procedures were analysed by electrophoresis in a 2% agarose gel containing ethidium bromide at 60 V for 4 hrs and were visualised by UV transillumination. The repeatability of the RAPD and BOX-PCR protocols were tested SIS3 by studying the isolates in three independent runs. DNA analysis The ISR and fliC gene sequences obtained were compared with sequences in the GenBank database using the Basic Local Alignment Search Tool (BLAST) [40] and aligned using the ClustalW program [41]. Phylogenetic and molecular evolutionary analyses were conducted using genetic distance based neighbour joining algorithms [42] within MEGA version 3.1 http://​www.​megasoftware.​net, [43]. The analysis of the RAPD and BOX gels was performed using BioNumerics software (version

5.1 Applied Maths, Kortrijk, Belgium), based on the Pearson Venetoclax in vitro correlation coefficient, and clustering by the unweighted pair group method with arithmetic means (UPGMA method) [44]. The isolates that clustered at a cut-off level of more than 80% similarity were grouped together; these were considered clonally related and classified into the same group. The discriminatory power of the BOX and RAPD-PCR for typing R. pickettii isolates was evaluated by using the discrimination index as described by Hunter and Gaston [30]. Accession numbers DNA sequences were deposited in the EMBL database with accession numbers for sequences from the 16S-23S spacer region are as follows: AM501933-AM501952 and for the FliC genes: FN869041-FN869057. Results Species-specific PCR To confirm that the isolates were in fact R.