In addition, the presence of very low-copy plasmids (fewer than five copies) may be constrained by this approach. Plasmid restriction analysis showed a diverse plasmid pool in intI+ strains from wastewaters. In total, 45 different plasmid restriction patterns (similarity < 98%) were obtained (Fig. 1). No restriction

patterns were recovered from six strains (MM.1.10, MM.1.11, MM.1.12, MM.1.14, MM.1.15, MM.1.26), due to low plasmid DNA concentration, which may be due to lower plasmid DNA extraction efficiency and/or very low-copy plasmid number. Restriction patterns did not cluster by species, type of effluent or treatment stage, suggesting a high diversity of backbones and/or accessory elements present in these strains. The results reinforce that wastewaters are reservoirs CHIR-99021 molecular weight of diverse mobile genetic elements and hotspots for HGT, as previously reported (Schlüter et al., 2007; Moura et al., 2010). Among donor strains, plasmids PCI-32765 cost were assigned to FrepB (Aeromonas salmonicida, Aeromonas veronii, Aeromonas sp., E. coli, Enterobacter sp.), FIC (A. salmonicida, Aeromonas sp.), FIA (Shigella sp.), I1 (A. veronii, Aeromonas

sp., E. coli), HI1 (E. coli) and U (Aeromonas media) replicons (Table 1 and Fig. 1). Although the presence of broad-host-range IncN, IncQ, IncW and IncP-1 plasmids had been detected in total community DNA obtained from the same environments (Moura G protein-coupled receptor kinase et al., 2010), none of

the donor strains gave positive hybridization signals using probes targeting these groups. Other studies dealing with total community DNA and exogenous isolation of plasmids from urban wastewaters also suggested that broad-host-range plasmids, in particular those belonging to the IncP-1 group, are abundant in wastewater environments (Dröge et al., 2000; Heuer et al., 2002; Schlüter et al., 2007; Bahl et al., 2009). Thus, results obtained here suggest that the hosts of broad-host-range plasmids may probably be noncultivable bacteria and/or bacteria from other taxa than those focused in this study. To date, reported replicons in Aeromonas spp. have been limited to IncU and IncA/C, identified in different aquatic environments. IncU replicons have been reported in Aeromonas caviae, A. media, Aeromonas allosaccharophila, Aeromonas hydrophila and A. salmonicida strains isolated from rivers (Cattoir et al., 2008), lakes (Picão et al., 2008), fish farms and hospital sewage (Rhodes et al., 2000), often associated with tetracycline and/or quinolone resistance determinants. IncA/C plasmids have been reported in A. hydrophila and A. veronii strains isolated from fish carriage water (Verner-Jeffreys et al., 2009).