8% agarose gel in 1X Tris/Borate/EDTA (TBE) buffer [18] and visualized to assess their integrity, then stored at 4°C prior to PCR amplification. BOX-PCR, ERIC-PCR and the molecular identification of selected Nutlin-3a chemical structure bacterial strains Amplification reactions using the primers BOXA1R [19] and ERIC1R and ERIC2F [20] were performed in the following mix: 1 μl (50–100 ng) of target DNA; 5 μl of 5X PCR buffer (Promega, RJ, Brazil); 2.5 mM (ERIC) or 3.75 mM (BOX) MgCl2; 0.5 mM dNTP; 0.4 μM

and 1 μM of the primers ERIC1R – ERIC2F or BOXA1R, respectively; and 0.5 U (ERIC) or 1.25 U (BOX) of Taq polymerase in a 25 μl final volume. The cycle applied was 1 × [7 min at 95°C], 35 × [1 min at 94°C, 1 min at 52°C (with ERIC primers) or 53°C (with BOXA1R primer), 8 min at 65°C] and a final extension of 16 min at 65°C. Negative controls (without DNA) were run during all amplifications. Agarose gel electrophoresis of PCR products was performed using 1.4% agarose in 1X TBE buffer at 90 V for 4 h at room temperature. The BOX and ERIC-PCR results were collected into matrices indicating the presence or absence (scored as 1 or 0, respectively) of bands. Dendrograms were constructed using Dice similarity coefficients and the unweighted pair group method with

arithmetic mean (UPGMA) through the BioNumerics software package (Applied Maths, Ghent, Belgium). For molecular identification find more of the selected isolates, their 16S rRNA coding gene was amplified by PCR using the pair of universal primers pA and pH and the

conditions described in Massol-Deya et al. [21]. The PCR products were then sequenced by Macrogen (South Korea). The partial 16S rRNA gene sequences (~800 bp) were identified using the BLAST-N tool (http://​blast.​ncbi.​nlm.​nih.​gov/​) on the National Center for Biotechnology Information (NCBI) website using the GenBank non-redundant database. A phylogenetic tree was constructed Silibinin based on partial 16S rRNA gene sequences using the neighbor-joining method. MEGA 5.1 software was used to calculate Jukes-Cantor distances. Bootstrap analyses were performed with 1,000 repetitions, and only values higher than 50% are shown in the phylogenetic tree. Susceptibility of the bacterial isolates to the essential oil obtained from L. sidoides genotypes LSID006 and LSID104 The determination of the minimum inhibitory concentration (MIC) was performed using a serial dilution technique in 0.2 ml thin-walled 8 strip cap microtubes as recommended by CLSI M7-A4 for bacteria [22]. Bacterial isolates from the four genotypes were tested for susceptibility. The investigated essential oils containing contrasting amounts of thymol and carvacrol (Table 1) were diluted seven times using doubling dilution, from 4 to 0.03 mg ml-1, and 1 μl of each dilution was added to 189 μl TSB with 10 μl of the bacterial suspension (cells grown to a O.D. = 0.09 at 625 nm, then diluted 50X in TSB). The microtubes were incubated for 48 h at 32°C.