The Antimicrobial Properties of Silver Nanoparticles in Bacillus subtilis Are Mediated by Released Ag+ Ions - PubMed (original) (raw)

The Antimicrobial Properties of Silver Nanoparticles in Bacillus subtilis Are Mediated by Released Ag+ Ions

Yi-Huang Hsueh et al. PLoS One. 2015.

Abstract

The superior antimicrobial properties of silver nanoparticles (Ag NPs) are well-documented, but the exact mechanisms underlying Ag-NP microbial toxicity remain the subject of intense debate. Here, we show that Ag-NP concentrations as low as 10 ppm exert significant toxicity against Bacillus subtilis, a beneficial bacterium ubiquitous in the soil. Growth arrest and chromosomal DNA degradation were observed, and flow cytometric quantification of propidium iodide (PI) staining also revealed that Ag-NP concentrations of 25 ppm and above increased membrane permeability. RedoxSensor content analysis and Phag-GFP expression analysis further indicated that reductase activity and cytosolic protein expression decreased in B. subtilis cells treated with 10-50 ppm of Ag NPs. We conducted X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses to directly clarify the valence and fine structure of Ag atoms in B. subtilis cells placed in contact with Ag NPs. The results confirmed the Ag species in Ag NP-treated B. subtilis cells as Ag2O, indicating that Ag-NP toxicity is likely mediated by released Ag+ ions from Ag NPs, which penetrate bacterial cells and are subsequently oxidized intracellularly to Ag2O. These findings provide conclusive evidence for the role of Ag+ ions in Ag-NP microbial toxicity, and suggest that the impact of inappropriately disposed Ag NPs to soil and water ecosystems may warrant further investigation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Morphology, particle size, and crystal structure characterization of Ag-NPs.

(A) SEM images of Ag NPs used in this study; white bar: 100 nm. (B) TEM images of Ag NPs used in this study; scale bar: 20 nm. (C) TEM image of a representative Ag NP from those used in this study; scale bar: 5 nm. (D) Size distribution histograms of Ag NPs derived from SEM analysis. (E) XRD patterns of synthesized Ag NPs.

Fig 2. Effects of various Ag-NP concentrations on B. subtilis growth.

(A) Growth analysis curves of B. subtilis in rich media treated with Ag NPs, measured by monitoring OD600 values. (B) Growth analysis curves of B. subtilis in minimal media treated with Ag NPs. Ag-NP concentrations are shown as -▢-: 0 ppm; -▷-: 0.1 ppm; -○-: 1 ppm; -△-: 5 ppm; -▽-: 10 ppm; -◇-: 25 ppm; and -◅-: 50 ppm. (C) Ag-NP antibacterial activity against B. subtilis cells.

Fig 3. Analysis of RedoxSensor content, PI staining, and Phag-GFP expression in B. subtilis.

(A) Wild-type bacteria were grown for 3 h with Ag-NP concentrations of 0, 5, 10, 25, or 50 ppm, and then subjected to RedoxSensorTM Green (green) and (B) PI (red) staining. PBS buffer-only and unstained controls were provided. The X axis indicates RedoxSensor or PI fluorescence intensity (arbitrary units: au), as measured by flow cytometry, and the Y axis indicates cell counts. The flow cytometry data is representative of two separate experiments. RedoxSensor activity presents a false green color, and PI presents a false red color. Scale bar: 10 μm. (C) B. subtilis Phag-GFP expression after treatment with varying concentrations of Ag NPs. Unstained controls were included. The X axis indicates GFP fluorescence intensity (arbitrary units: au), and the Y axis indicates cell counts. Flow cytometry data is representative of two separate experiments. (D) Fluorescent micrographs indicate Phag-GFP expression in B. subtilis cells cultivated with various concentrations of Ag NPs for 3 h. GFP reporter expression presents as a false green color. DAPI presents a false blue color. Scale bar: 10 μm.

Fig 4. K-edge XANES spectra of silver standards and Ag NP-treated B. subtilis cells.

(A) Normalized Ag K-edge XANES spectra. (B) Derivative Ag K-edge XANES spectra. The spectra are displaced vertically for clarity.

Fig 5. K-edge EXAFS oscillation k3χ(k) of silver standards and Ag NP-treated B. subtilis cells.

Spectra from Ag NP-treated B. subtilis cells were fitted to spectra from (A) Ag2O, (B) Ag2S, (C) AgO, and (D) Ag standards. (E) Ag Fourier transformation (FT) spectra of silver standards and B. subtilis cells treated with 100 ppm of Ag NPs.

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The Antimicrobial Properties of Silver Nanoparticles in Bacillus subtilis Are Mediated by Released Ag+ Ions - PubMed (original) (raw)