Figure 1.
Characterization of silver nanoparticles.
A: Transmission electron microscopy images of AgNPs. TEM image of collargol is reprinted from [21] with the permission of Springer. B: Stability of 10 mg/l nanoparticles suspensions in test medium (half-strength NaCl-free LB medium). After 0-h incubation (upper panel), all AgNPs suspensions scattered light upon the illumination by the laser pointer, indicating the presence of particles. After 4-h incubation (lower panel), the light scattering from uncoated nAg was negligible (comparable to that of the test medium with no AgNPs added) and settling of the particles was observed.
Table 1.
Characterization of silver nanoparticles (AgNPs) used in the current study.
Figure 2.
4-h EC50 values (endpoint: growth inhibition; note the logarithmic scale) for different silver formulations to bacteria.
nAg-PVP states for PVP-coated AgNPs and nAg-Col for collargol. Concentrations are nominal. Data represent the average ± SD (n = 3), p-values denote statistically significant differences between different bacteria (ANOVA).
Figure 3.
Response of Escherichia coli MC1061 to silver formulations.
A: Dose-dependent induction of bioluminescence of Ag-biosensor E. coli MC1061 (pSLcueR/pDNPcopAlux) in response to silver formulations. The bioluminescence was measured after 4-h exposure (30°C, half-strength NaCl-free LB medium). The decreased bioluminescence at higher concentrations refers to the toxicity. Concentrations are nominal. B: Toxicity (4-h EC50) of silver nanoparticles and AgNO3 to E. coli MC1061. EC50 values based on nominal concentrations (open columns) are from Figure 2; EC50 values based on intracellular Ag ions (red columns) are re-calculated from Figure 3A. Data represent the average ± SD (n = 3).
Figure 4.
Schematic representation of the experiment to study dissolution of AgNPs.
Intracellular Ag ions, extracellular dissolved Ag and extracellular Ag ions were quantified in biotic (left) as well abiotic (right) conditions. Ag-biosensor E. coli MC1061(pSLcueR/pDNPcopAlux) was exposed either to the 10 mg/l suspensions of AgNPs (biotic dissolution) or to the supernatants obtained after ultracentrifugation (390 000 g×60 min) of the respective AgNPs' suspensions (abiotic dissolution). Intracellular Ag ions were quantified as a function of increase of bioluminescence Ag-biosensor E. coli MC1061(pSLcueR/pDNPcopAlux), extracellular dissolved Ag was measured by atomic absorption spectroscopy (AAS) and extracellular Ag ions by ion-selective electrode (Ag-ISE).
Figure 5.
Dissolved, free and intracellular concentrations of Ag in biotic and abiotic conditions.
A: Dissolved, free and intracellular Ag after 4-h direct cell-NP contact. Intracellular Ag+ was quantified from bioluminescent response of E. coli MC1061 (pSLcueR/pDNPcopAlux) to AgNPs suspension. Extracellular dissolved Ag was measured by atomic absorption spectroscopy (AAS) and free Ag+ was measured by Ag-ion-selective electrode (Ag-ISE) from the test medium after settling of NPs and bacterial cells by ultracentrifugation at 390 000 g for 1 hour. B: Dissolved, free and intracellular Ag in abiotic conditions. Extracellular dissolved Ag (AAS), extracellular free Ag+ (Ag-ISE) and intracellular Ag+ (Ag-biosensor E. coli MC1061 (pSLcueR/pDNPcopAlux)) were measured from the test medium after settling of NPs by ultracentrifugation at 390 000 g for 1 hour. Data represent the average ± SD (n = 3). **p<0.01, ns – not significant (p>0.05) compared to abiotic dissolution according to two-tailed unpaired t-test.
Figure 6.
Bacterial growth after 4-h exposure to collargol directly and through the dialysis membrane.
Escherichia coli MC1061 (A, C) and Pseudomonas aeruginosa DS10-129 (B, D) upon exposure to collargol (nAg-Col; A, B) after 4 h in half-strength NaCl-free LB at 30°C in the direct contact with nanoparticles (rectangle) or being separated from particles by 20 kDa (4 nm) cut-off membrane (diamond). AgNO3 was handled analogously to collargol and was used as a control (C, D). Data represent the average ± SD (n = 4).
Figure 7.
UV-visible absorption spectra of nAg-Col suspensions (10 mg Ag/l) with or without bacteria.
UV-Vis spectra of AgNPs in half-strength NaCl-free LB medium without bacterial cells (dashed red line) or after exposure of bacterial cells to nAg-Col (solid line). Before measurement, bacterial cells and cell-associated nanoparticles were removed by centrifugation at 4 000 g for 5 minutes. The difference between dashed and continuous lines shows the co-precipitation of nAg-Col particles with bacterial cells. The representative figures from the three independent experiments are shown.