Analysis of the Bacterial Vesicles' Enhanced Toxicological Threat Via Electron Microscopy

Bacterial Biofilm, Plastic Biodestruction, And Nanoparticles Formation

Staphylococcus aureus is a Gram-positive bacterium permanently present in the human body as a commensal, and often as a pathogen as well (Lowy, 1998). When polyurethane prosthetic devices come into contact with S. aureus, its ability to colonize medical implants, adhering to the polymeric surface and forming a biofilm, results in the development of a community with dynamics able to highly affect the material’s stability. The biofilm formation is a four-steps process (Figure 1) where the bacterial initial adhesion onto the polymer is followed by the formation of microcolonies, by the maturation phase with the appearance of the exopolysaccharide matrix and eventually by the detachment of nomadic cells (Didenko et al., 2012; Didenko et al., 2013; Curia et al., 2014; Ghannoum & O'Toole, 2004).

The action of a bacterial biofilm, characterized by high cell density and limitation of nutrients (Fux, Costerton, Stewart, & Stoodley, 2005), provokes damages on the prosthesis and causes the detachment of debris (micro- and nano-particles) from the bulk material (biodestruction) (Didenko et al., 2012; Curia et al., 2014) that will be well documented by scanning and transmission electron microscopy techniques (Figures 2, 3, and 4).

Figure 2.

SEM (Scanning Electron Microscope) image of a S. aureus biofilm on a polyurethane surface after a 45-days-long incubation. It is evident the presence of the exopolysaccharide matrix. Polyurethane nanoparticles are detectable throughout the whole biofilm embedded in the matrix (circles).

Figure 3.

STEM Dark Field image of a portion of a S. aureus cell. Bright polyurethane nanoparticles are visible in the external medium, on the cell wall and inside the bacterium. A trail of vesicles (arrows) from the periphery to the center of the cell is detectable. The cell wall is undergoing a modification phase, and on the left it is visible that components of the peptidoglycan cell wall are dispersing in the extracellular environment. Vesicles exiting the bacterial cell are visible by the scaling of the cell wall (circle).

Figure 4.

magnification of a membranous vesicle inside a S. aureus cell. In this TEM Bright Field image, electron dense polyurethane nanoparticles (2-3 nm) appear darker than the surrounding components. Vesicle diameter measures 30 nm. Horizontal Field Width (HFW) = 65 nm. The thickness of the vesicle’s plasma membrane is of about 4-5 nm.

Figure 1.

Scheme of the formation of a coccal biofilm and polyurethane biodestruction

1. Initial adhesion of S. aureus cells to the polyurethane surface;2. Formation of microcolonies;3. Biofilm maturation, appearance of the exopolysaccharide matrix, initial biodestruction of polyurethane and absorption of polymeric nanoparticles by bacterial cells;4. Detachment of nomadic cells loaded with polyurethane nanoparticles from the mature biofilm.