Outer Membrane Vesicles (OMV) preparation was performed following the protocol reported for A. hydrophila [49] (link). A. trota with the aphA chromosomal resistance marker, A. caviae 6548 and E. coli 10G, both carrying pRANGER-BTB3 plasmid, were used as vesicle donors. The presence of DNA inside DNase-treated OMV was assessed by extracting whole genomic DNA as previously described [44] . PCR was carried out to confirm the presence of the aphA gene and pRANGER BTB-3 inside the vesicles (Table S1). Vesicles electron microscopy was done as follows. OMV suspensions were placed on copper grids coated with formvar and dried using filter paper. Samples were left in contact with the grid for one minute. An amount of 2.5% uranyl acetate was added and left in contact for another minute, then it was removed and the grid was left to dry at room temperature. Vesicles were observed under transmission electron microscopy (Zeiss model Libra 120, Oberkochen, Baden-Württenberg, Germany). Images were digitalized with Gatan Digital Micrograph software v3.
DNA transfer via OMV was carried out as described by Rumbo et al. [50] (link). After contact between OMV and receptor cells, A. trota transformants were selected on Luria agar plates supplemented with the appropriate antibiotic. E. coli JM109 was used as a positive control [51] (link).
DNA transfer via OMV was carried out as described by Rumbo et al. [50] (link). After contact between OMV and receptor cells, A. trota transformants were selected on Luria agar plates supplemented with the appropriate antibiotic. E. coli JM109 was used as a positive control [51] (link).