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.Together with Pseudomonas aeruginosa, Burkholderia cepacia is a primary opportunistic pathogen of cystic fibrosis patients. Infection with B. cepacia is generally associated with aggressive necrotizing pneumonia and is accompanied by an acute systemic infection, such as bacteremia or septicemia (8). This rapid clinical decline due to B. cepacia colonization is known as the so-called "cepacia syndrome" and leads to mortality in 20 to 35% of chronically infected individuals (9). Treatment is often made more difficult due to the innate impermeability of B. cepacia's outer membrane (OM) to antibiotics like aminoglycosides, polymyxin, and ␤-lactams (7,17,25).One potential method of circumventing this resistance is through the use of a specific membrane-based antibiotic delivery system, such as gentamicin-containing membrane vesicles (g-MVs) that can breach the OM. The g-MVs from P. aeruginosa PAO1 successfully deliver gentamicin and a peptidoglycan hydrolase into both gram-positive and -negative bacteria (11,12,16). For gram-negative pathogens, g-MVs contact the bacterium's OM and fuse into it so as to release the vesicle's contents into the periplasm of the cell. Here, the (now) periplasmic gentamicin is actively taken into the cytoplasm to inhibit protein synthesis. At the same time, the (now) periplasmic peptidoglycan hydrolase begins to hydrolyze the host's peptidoglycan layer. This two-pronged attack by the g-MVs might be an attractive system to use against pathogens that are intrinsically impermeable to antibiotics, especially since g-MVs are thermodynamically stable and do not break down in suspension (12, 16). The aim of the present work was to study the utility of g-MVs against B. cepacia. Strains C5424 and CEP0248 were chosen as the test strains because they represent members of the B. cepacia group IIIa complex, which comprises 80% of the B. cepacia clinical isolates in Canada (22). Additionally, these two distinct strains possess smooth lipopolysaccharide (LPS) and cable (Cbl) pili, suggesting that they might present similar surfaces for g-MV attachment. The striking difference between the strains is in their susceptibility to gentamicin. The MIC of gentamicin for CEP0248 is 5 g/ml, while that for C5424 is 20 g/ml.The g-MVs were generated from P. aeruginosa PAO1 as described previously (12) and contained 7.0 (Ϯ1.0) ng of gentamicin/g of MV protein as estimated by enzyme-linked immunosorbent assay (12, 16).

Extracellular Vesicles: An Overlooked Secretion System in Cyanobacteria

“… Diagram summarizing the biological roles proposed for extracellular vesicles from Gram-negative bacteria. EVs may have the following functions: in infection, by eluding the host immune response; as public goods, as they may carry catalytic enzymes that help deconstructing complex extracellular biomolecules, making them available for the EVs-releasing bacteria and for other organisms; in horizontal gene transfer, as they transport nucleic acids that can be transferred between organisms; in communication, e.g., in the context of biofilms; in the uptake of nutrients (e.g., [ 96 , 97 ]); as decoy, for example to evade phage infection; and in the secretion of antibiotics (e.g., [ 98 , 99 ]). …”

Prokaryotic membrane vesicles: new insights on biogenesis and biological roles

“…An increase in OMV production after exposing the bacterial cells to toxic compounds such as antibiotics, stressor substances, phages or the host environment at the infection site have been observed, probably constituting a new defense mechanism (Loeb, 1974;Grenier and Belanger, 1991;Allan and Beveridge, 2003;Nevot et al, 2006;Irazoqui et al, 2010). Under these harsh conditions, bacterial cells were able to secrete unnecessary or unwanted material, such as unfolded or over-expressed proteins, to reduce the envelope stress ( Figure 1).…”

Virulence Factors of the Oral Spirochete Treponema denticola

“…OMVs are considered potent virulence factors, since they possess adhesins, toxins, and proteolytic enzymes, can mediate bacterial aggregation and invasion, are cytotoxic, and can modulate the host immune response (Kuehn and Kesty, 2005). Besides affecting the host, OMVs may be important for securing a niche in the competitive environment of subgingival plaque by eliminating competitors via the delivery of proteases and toxins (Kadurugamuwa and Beveridge, 1996;Allan and Beveridge, 2003). OMVs also facilitate the remote delivery of labile signaling molecules and prevent their degradation by other microorganisms (Z Li et al, 1996;Mashburn and Whiteley, 2005).…”