Phenotypic characters were scored as discrete variables [0 or 1]; 0, when the character was negative or missing; 1, when character selleck chemical was positive or present). Isolates with the same pattern were grouped into Biotypes numbering 1 to 35. The unweighted pair group method

[28] was used for cluster analysis using the MultiVariate Statistical Package (MVSP) software program ver. 3.13 by means of the Jaccard coefficient [29]. The discriminatory power of the biotyping for typing R. pickettii isolates was evaluated by using the discrimination index as described by Hunter and Gaston, as given by the equation: D = 1 – [1/N (N - 1)] ∑nj (nj – 1), where D is the numerical index of discrimination, N is the total number of isolates, and nj is the number of isolates pertaining to the jth type [30]. Genotypic analysis DNA for all PCR experiments was prepared as described previously [31]. Species-specific PCR and amplification 16S-23S rRNA ISR and fliC gene The species-specific PCR primers (Rp-F1, Rp-R1 and R38R1) used in this study were designed by Coenye et al., detailed in Table 2[32, 33]. The 16SF and 23SR primers were used to amplify the Interspacial Region selleck (ISR) [34] and

the Ral_fliC primers (Ral_fliCF and Ral_fliCR) were used to amplify the fliC gene (Table 2), [35]. The PCR assays were performed in 25 μL reaction mixtures, containing 100 ng of template genomic DNA, 1U Taq polymerase,

250 mM (each) deoxynucleotide triphosphate, 1.5 mM MgCl2, 10x PCR buffer (Bioline), and 20 pmol of oligonucleotide primer (MWG Biotech, Ebersberg, Germany) Rp-F1 and 10 pmol of oligonucleotide primers Rp-R1 and R38R1 for the species-specific PCR and 20 pmol each of the primers for the ISR and fliC regions (Table 2). After initial denaturation for 2 min at 94°C, 30 amplification cycles were completed, each consisting click here of 1 min at 94°C, 1 min at 55°C, and 1 min 30 secs at 72°C. A final extension of 10 min at 72°C was then applied. The PCR products were analysed by electrophoresis in a 1.5% agarose gel (Agarose MP, Roche Diagnostics) for 1 hour (100 V) with ethidium bromide staining in the TBE buffer and photographed under the UV light (UV Products Gel Documentation System Imagestore, Ultra Violet Products, Cambridge). A 200-10000bp DNA ladder (Bioline) was included on all gels to allow standardization and sizing. Following amplification of the ISR and fliC region from test isolates PCR product was purified using the NucleoSpin Extract II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions and the amplicons sequenced (MWG Comfort Read service).