Gentamicin delivery to Burkholderia cepacia group IIIa strains via membrane vesicles from Pseudomonas aeruginosa PAO1

Abstract

When Pseudomonas aeruginosa PAO1 is treated with gentamicin, it releases membrane vesicles containing gentamicin (g-MVs) and peptidoglycan hydrolase, which makes the MVs bactericidal. We evaluate the ability of g-MVs to deliver gentamicin past the intrinsic permeability barrier of group IIIa Burkholderia cepacia and show that strain CEP0248 with low resistance to gentamicin is killed but the highly resistant strain C5424 is not. Immunoelectron microscopy revealed that gentamicin was delivered into both strains, suggesting that there might be another mechanism of resistance in C5424.

FIG. 1.

Bactericidal assays to monitor the effects on B. cepacia C5424 (A) and CEP0248 (B) of soluble gentamicin at 2.5 times the MIC (×), PAO1 n-MVs (▪), PAO1 g-MVs (▴), and a HEPES buffer control (⧫). The MVs were added at a protein concentration of 100 μg/ml, and error bars represent standard deviations.

FIG. 2.

Negatively stained whole mount of C5424 showing the delicate Cbl pili that are removed by washing during the immunogold labeling described in the legend to Fig. 3. Scale bar, 1 μm.

FIG. 3.

TEM images of negatively stained C5424 cells labeled with gold-conjugated anti-B MAbs towards PAO1 LPS. (A) Image from the antibactericidal assay at time zero. Arrows point to labeled MVs in the process of attaching to a cell. (B) Image taken at 1 h. Arrows show labeled MVs. (C) Image taken at 24 h. The gold label can be seen throughout the surface of this cell, indicating that the PAO1 g-MVs have fused the membrane and the PAO1 LPS has spread over the cell surface. (D) Control cells labeled with anti-B MAbs but without PAO1 g-MVs added. Scale bars, 100 nm.

FIG. 4.

Thin sections of C5424 labeled with gold-conjugated antibodies to gentamicin. (A) Control treated with n-MVs from PAO1; (B and C) g-MVs fusing to the OM. Small arrows show MVs containing gentamicin, whereas the large arrow in panel C shows gentamicin within the cytoplasm. Scale bars, 100 nm.