Putative transconjugants were confirmed by BOX-PCR typing. Profiles were generated by PCR amplification in 25 μL reaction mixtures containing 3.75 mm MgCl2, 0.2 mm dNTPs, 1× Stoffel buffer, 0.2 μm of primer BOX-AIR (5′-CTACGGCAAGGCGACGCTGACG-3′; Versalovic et al., 1991), 2.5 U Stoffel Taq polymerase (Applied Biosystems) and 1 μL of cell suspension prepared in 100 μL of distilled Dasatinib manufacturer water (~ 1.0 McFarland turbidity standard). Amplification was carried out as follows: initial denaturation for

7 min at 94 °C, then 35 cycles of denaturation at 94 °C for 7 min, followed by annealing at 53 °C for 1 min and extension at 65 °C for 8 min, and a final extension at 65 °C for 16 min. Generated profiles were separated in 1.5% agarose gels in 0.5× TBE buffer (50 mm Tris, 50 mm boric acid, 0.5 mm EDTA), at 50 V for 95 min, and stained with ethidium bromide. Plasmid DNA from donors and transconjugants was purified using Qiagen Plasmid Mini-kit (Qiagen GmbH, Germany). Diversity of plasmids was evaluated by plasmid restriction analysis using 5 U of PstI (CTGCAG) and 5 U of Bst1770I (GTATAC), according to the manufacturer’s instructions (Fermentas, Lithuania). Restriction patterns were visualized in 0.8% agarose gels. Electrophoresis was run at 40 V for 3 h in 0.5× TBE buffer and stained using ethidium bromide. Restriction

PI3K assay patterns were compared using GelCompar II software (Applied Maths, SintMartens-Latem, Belgium). Detection 3-oxoacyl-(acyl-carrier-protein) reductase of IncP-1, IncQ, IncN and IncW replicons and integrase genes was performed as previously described (Götz et al., 1996; Moura et al., 2010). Briefly, gels were transferred onto nylon membranes (Hybond-N, Amersham,

Germany) and hybridized in middle stringency conditions with PCR-derived specific digoxigenin-labelled probes for intI1, intI2, IncP-1 (trfA), IncQ (oriV), IncN (rep) and IncW (oriV) (Moura et al., 2010). Detection of IncA/C, IncB/O, IncF (FIA, FIB, FIC, FIIA, FrepB subgroups), IncHI1, IncHI2, IncI1-Iγ, IncK, IncL/M, IncU, IncT and IncY replicons was performed by PCR, using primers and conditions previously described (Carattoli et al., 2005). Results were confirmed by sequencing, except for IncFrep replicons, which were confirmed by Southern hybridization with digoxigenin-labelled probes generated by PCR from positive controls (Carattoli et al., 2005). The aim of this study was to evaluate the occurrence, diversity and conjugative potential of plasmids in integron-carrying bacteria from wastewater environments. The presence of plasmid DNA was confirmed in 77% (51 out of 66) of the strains. In the remaining 15 strains (~ 23%), no plasmids were detected by the plasmid extraction method used. Thus, most of the strains analysed harboured at least one plasmid, these strains being retrieved from all stages of the treatment process, including from final effluents (Table 1). Nevertheless, the presence of additional plasmids cannot be excluded.