Exploring Antibacterial Activity and Bacterial-Mediated Allotropic Transition of Differentially Coated Selenium Nanoparticles

Abstract

The use of metal nanoparticles (NPs) as antimicrobial agents has become a promising alternative to the problem of antibiotic-resistant bacteria and other applications. Silver nanoparticles (AgNPs) are well-known as one of the most universal biocide compounds. However, selenium nanoparticles (SeNPs) recently gained more attention as effective antimicrobial agents. This study aims to investigate the antibacterial activity of SeNPs with different surface coatings (BSA-coated, chitosan-coated, and undefined coating) on the Gram-negative Stenotrophomonas bentonitica and the Gram-positive Lysinibacillus sphaericus in comparison to AgNPs. The tested NPs had similar properties, including shape (spheres), structure (amorphous), and size (50-90 nm), but differed in their surface charge. Chitosan SeNPs exhibited a positive surface charge, while the remaining NPs assayed had a negative surface charge. We have found that cell growth and viability of both bacteria were negatively affected in the presence of the NPs, as indicated by microcalorimetry and flow cytometry. Specifically, undefined coating SeNPs displayed the highest percentage values of dead cells for both bacteria (85-91%). An increase in reactive oxygen species (ROS) production was also detected. Chitosan-coated and undefined SeNPs caused the highest amount of ROS (299.7 and 289% over untreated controls) for S. bentonitica and L. sphaericus, respectively. Based on DNA degradation levels, undefined-SeNPs were found to be the most hazardous, causing nearly 80% DNA degradation. Finally, electron microscopy revealed the ability of the cells to transform the different SeNP types (amorphous) to crystalline SeNPs (trigonal/monoclinical Se), which could have environmentally positive implications for bioremediation purposes and provide a novel green method for the formation of crystalline SeNPs. The results obtained herein demonstrate the promising potential of SeNPs for their use in medicine as antimicrobial agents, and we propose S. bentonitica and L. sphaericus as candidates for new bioremediation strategies and NP synthesis with potential applications in many fields.

Keywords: antibiotic; applications; bioremediation; nanoparticles; selenium.

Figure 1

Maximum metabolic activity of L. sphaericus and S. bentonitica treated with 100 μM metal NP, normalized by an untreated control. The maximum metabolic activity was determined between 2 and 8 h after treatment and corresponds to the maximum heat flow, measured by microcalorimetry. Ordinary one-way ANOVA statistical test was performed with α = 0.05. ns represents non-significant (p > 0.05); *** represents p < 0.0005.

Figure 2

Ratio of dead cells over time of L. sphaericus (A) and S. bentonitica (B) in contact with the tested NPs relative to the respective untreated control (NP concentration: 100 μM). ROS content generated by L. sphaericus (C) and S. bentonitica (D) after 3 h’s exposure with NPs at different concentrations (1, 10, 50, and 100 μM). Membrane potential as a measure of membrane polarization over time of L. sphaericus (E) and S. bentonitica (F) after exposure to the tested NPs at 100 μM concentration. The intracellular DNA content of L. sphaericus (G) and S. bentonitica (H) cells after 48 h in contact with the tested NPs at different concentrations (1, 10, 50, and 100 μM). Tested NPs: AgNPs (gray), BSA-coated SeNPs (blue), UD-SeNPs (undefined coating in red), and chitosan-coated (CS-SeNPs in green).

Figure 3

Environmental scanning electron microscopy micrographs of L. sphaericus (left panels: A,C,E) and S. bentonitica (right panels: B,D,F) incubated for 24 h with 100 μM UD-SeNPs (A,B), BSA-SeNPs (C,D), and CS-SeNPs (E,F), respectively. Elemental composition of the NPs was investigated by EDX (see the insets). Additional SeNPs interacting with the bacterial cells are highlighted by arrows. Scale bars: A (4 μm), B–E (3 μm), and F (2 μm).

Figure 4

Electron microscopy micrographs of thin sections of L. sphaericus treated with 100 μM UD-SeNPs (A–D), BSA-SeNPs (E–H), and CS-SeNPs (I–L) for 24 h, respectively. The dashed boxes highlight the areas which were analyzed by EDX-element mapping, with the results displayed for selenium in red, phosphorus in yellow green, and sulfur in cyan. Scale bars: 700 nm (A–D), 500 nm (E–I), and 200 nm (J–L).

Figure 5

HAADF-STEM images showing selenium nano- and microstructures (A,C) after exposure of L. sphaericus to 100 μM UD-SeNPs for 24 h. HR-TEM micrographs (B,D) corresponding to regions 1 and 3, and SAED patterns (E,F) corresponding to regions 2 and 4. Scale bars: A and C (100 nm) and B and D (5 nm).