Plasma membrane is the target of rapid antibacterial action of silver nanoparticles in Escherichia coli and Pseudomonas aeruginosa

This work was supported by Estonian Research Council grants IUT23-5 and PUT1015 and by Research Council of Lithuania, funding grant no MIP-040/2015Silver nanoparticles (AgNP) are widely used in consumer products and in medicine, mostly due to their excellent antimicrobial properties. One of the generally accepted antibacterial mechanisms of AgNP is their efficient contact with cells and dissolution in the close vicinity of bacterial cell envelope. Yet, the primary mechanism of cell wall damage and the events essential for bactericidal action of AgNP are not elucidated. Materials and methods: In this study we used a combination of various assays to differentiate the adverse effects of AgNP on bacterial cell envelope: outer membrane (OM) and plasma membrane (PM). Results: We showed that PM was the main target of AgNP in gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: AgNP depolarized PM, induced the leakage of the intracellular K+, and inhibited cellular respiration. The results of bacterial bioluminescence inhibition assay in combination with AgNP dissolution and oxidation assays demonstrated that the adverse effects of AgNP occurred at concentrations 7–160 µM. These toxic effects occurred already within the first few seconds of contact of bacteria and AgNP and were driven by dissolved Ag+ ions targeting bacterial PM. However, the irreversible inhibition of bacterial growth detected after 1-hour exposure occurred at 40 µM AgNP for P. aeruginosa and at 320 µM AgNP for E. coli. In contrast to effects on PM, AgNP and Ag+ ions had no significant effect on the permeability and integrity of bacterial OM, implying that AgNP indeed targeted mainly PM via dissolved Ag+ ions. Conclusion: AgNP exhibited antibacterial properties via rapid release of Ag+ ions targeting the PM and not the OM of gram-negative bacteriaBiochemijos katedraGamtos mokslų fakultetasVytauto Didžiojo universiteta

Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID

<p>Within EU FP7 project NANOVALID, the (eco)toxicity of 7 well-characterized engineered nanomaterials (NMs) was evaluated by 15 bioassays in 4 laboratories. The highest tested nominal concentration of NMs was 100 mg/l. The panel of the bioassays yielded the following toxicity order: Ag > ZnO > CuO > TiO₂>MWCNTs > SiO₂>Au. Ag, ZnO and CuO proved very toxic in the majority of assays, assumingly due to dissolution. The latter was supported by the parallel analysis of the toxicity of respective soluble metal salts. The most sensitive tests/species were <i>Daphnia magna</i> (towards Ag NMs, 24-h EC₅₀=0.003 mg Ag/l), algae <i>Raphidocelis subcapitata</i> (ZnO and CuO, 72-h EC₅₀=0.14 mg Zn/l and 0.7 mg Cu/l, respectively) and murine fibroblasts BALB/3T3 (CuO, 48-h EC₅₀=0.7 mg Cu/l). MWCNTs showed toxicity only towards rat alveolar macrophages (EC₅₀=15.3 mg/l) assumingly due to high aspect ratio and TiO₂ towards <i>R. subcapitata</i> (EC₅₀=6.8 mg Ti/l) due to agglomeration of TiO₂ and entrapment of algal cells. Finally, we constructed a decision tree to select the bioassays for hazard ranking of NMs. For NM testing, we recommend a multitrophic suite of 4 <i>in vitro</i> (eco)toxicity assays: 48-h <i>D. magna</i> immobilization (OECD202), 72-h <i>R. subcapitata</i> growth inhibition (OECD201), 30-min <i>Vibrio fischeri</i> bioluminescence inhibition (ISO2010) and 48-h murine fibroblast BALB/3T3 neutral red uptake <i>in vitro</i> (OECD129) representing crustaceans, algae, bacteria and mammalian cells, respectively. Notably, our results showed that these assays, standardized for toxicity evaluation of “regular” chemicals, proved efficient also for shortlisting of hazardous NMs. Additional assays are recommended for immunotoxicity evaluation of high aspect ratio NMs (such as MWCNTs).</p