, 2010). Although integrons are transposition defective, they can be mobilized in association with functional transposons and/or conjugative plasmids (Cambray et al., 2010). Despite their relevance in HGT processes, the association of integrons with conjugative plasmids has been poorly addressed in aquatic environments. Wastewater treatment plants (WWTPs) are important reservoirs of resistance determinants and favourable places for HGT, due to high microbial abundance, high nutrient concentrations and intense selective pressures imposed by antibiotics, detergents and other pollutants

(Schlüter et al., 2007). Moreover, it has been shown that natural conjugative plasmids may induce the development of biofilms, which might also increase the chances of cell-to-cell contact and the occurrence of HGT events (Ghigo, 2001). As a result, WWTPs may favour the U0126 purchase persistence of plasmids through the treatment Anti-diabetic Compound Library chemical structure process, contributing to the dissemination

of integrons and undesirable genetic traits, such as those coding for antibiotic resistance and virulence determinants, to natural waters, soils and eventually the food chain. Previously, the presence and distribution of integron-carrying bacteria was investigated at different stages of the treatment process in two WWTPs, one treating urban discharges and the other treating wastewaters from a slaughterhouse (Moura et al., 2007, 2012). The present study was performed DOK2 to investigate the diversity of plasmids in integron-positive strains retrieved from wastewaters, providing data pertaining to the contribution of these environments to the spread of integrons and antibiotic resistance determinants through HGT. Sixty-six integron-positive (intI+) strains belonging to Aeromonas sp. (n = 48) and Enterobacteriaceae (n = 18) previously isolated from urban and slaughterhouse wastewaters (Moura et al., 2007, 2012) were included as donors in mating assays using rifampicin- and kanamycin-resistant Escherichia coli CV601-GFP

and Pseudomonas putida KT2442-GFP as recipient strains (Smalla et al., 2006). Liquid cultures of donor and recipient strains were prepared separately in 10 mL Luria–Bertani broth (LB) and grown overnight with gentle shaking at 28 °C. Recipient and donor strains were mixed (ratio 1 : 1) and centrifuged for 5 min at 6700 g to precipitate cells. Supernatants were discarded and replaced by 1 mL fresh LB. Mixtures were incubated overnight at 28 °C without shaking. Cells were then precipitated by centrifugation (5 min, 6700 g) and washed in 0.9% NaCl solution. Serial dilutions were prepared in 0.9% NaCl and aliquots of 100 μL were spread on Plate Count Agar plates supplemented with rifampicin (50 mg L−1) and streptomycin (50 mg L−1) or with rifampicin (50 mg L−1) and tetracycline (50 mg L−1). Putative transconjugants were grown at 28 °C for 48 h. Assays were run in duplicate.