FIG 4.

Biofilm-derived P. aeruginosa PAO9503 OMVs package plasmid DNA encoding antibiotic resistance. (A) Control and DNase-treated P. aeruginosa PAO9503 pBBR1MCS-5 planktonic-derived OMVs (pOMVs) and biofilm-derived OMVs (bOMVs) were stained with the lipophilic stain DiO (green) and the membrane-permeable DNA stain SYTO-61 (red) to observe DNA associated with OMVs and examined using confocal microscopy. Red boxes show the area that is magnified from the merged images (zoom). Areas of colocalization are shown in white and are indicated by white arrows. Images are representative of n = 3 biological replicates. Scale bar, 10 μm. (B) DNA associated with P. aeruginosa PAO9503 pBBR1MCS-5 control (−; filled shapes) and DNase-treated (+; open shapes) pOMVs (circles) and bOMVs (squares) was quantified using Qubit and is presented in nanograms of DNA per 10⁸ OMVs. pBBR1MCS-5 plasmid DNA was used as a DNase control (plasmid DNase control; triangles). Data shown are means ± SEM of n ≥ 3 biological replicates. ns, not significant; *, P < 0.05; ****, P < 0.0001 (Student’s t tests). (C) PCR of control (control bOMVs) or DNase-treated bOMVs (bOMVs + DNase) isolated from biofilm cultures of PAO9503 pBBR1MCS-5 using primers specific for the gentamicin resistance cassette (Gm^r). H₂O was used as a negative control (PCR − control) and pBBR1MCS-5 was used as a positive control (PCR + control). Arrow represents the gentamicin cassette amplicon (Gm^r, 500 bp). Image is representative of n = 3 biological replicates. (D) The relative copy number of the Gm^r gene from pBBR1MCS-5, as a measure of plasmid copy number, was calculated per 10⁸ OMVs obtained from planktonic PAO9503 pBBR1MCS-5 (pOMVs; circles) or PAO9503 pBBR1MCS-5 biofilm cultures (bOMVs; squares). OMVs were left untreated (−; filled shapes) or DNase treated (+; open shapes) prior to plasmid quantification by qPCR. Data are means ± SEM of n = 5 biological replicates. ns, not significant; *, P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons).