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- Materials and methods
The unique properties of metal nanoparticles allow them to have great potential in research and development and diverse applications in industrial products (Caruthers et al. 2007; Park 2007; Engel et al. 2008; Theron et al. 2008; Banerjee et al. 2011; Duncan 2011). The high surface-to-volume ratio of nanoscale materials is associated with a number of novel and desirable properties compared with the corresponding bulk materials. These properties include chemical, mechanical, electrical and optical characteristics such as light absorption and conductivity, as well as catalytic and biological activity (Park 2007; Nel et al. 2009). In particular, the vigorous antimicrobial properties of nanoscale metal and metal oxide particles such as Ag, TiO2 and ZnO have been the focus of industrial applications in biocidal coatings (e.g. filters for air and water treatment, clothes and other textiles, paints and varnishes, cosmetics and personal care products).
Silver, especially in its nanoscale form, has a strong toxicity towards a wide range of micro-organisms. Because of their large surface area, Ag(0) nanoparticles also release bioactive silver ions more effectively than bulk Ag(0). This biocidal property allows for the prevention and topical treatment of infectious diseases and also the production of antimicrobial, self-cleaning and self-disinfectant surfaces. Such applications of Ag(0) nanoparticles can be found in fillers and coatings in medical devices, implant and prosthetic materials and health care products (e.g. Schneider et al. 2008; Eby et al. 2009; Stevens et al. 2009; Lara et al. 2011; Taylor and Webster 2011; Zhao et al. 2011). Because the same biocidal effect can be achieved with relatively small input of raw materials, nanoparticles contribute to an efficient use of materials (Nel et al. 2006; Lok et al. 2007; Martinez-Castanon et al. 2008; Liu et al. 2010; Dal Lago et al. 2011).
The extremely high reactivity of metal nanoparticles is associated both with known and unknown toxic effects, including those against micro-organisms (Klaine et al. 2008; Nel et al. 2009; Marambio-Jones and Hoek 2010). A prerequisite for understanding the cellular mechanisms of the antimicrobial effects is to monitor the susceptibility of micro-organisms to biocidal metal nanoparticles. Such metal nanoparticles interact with microbial cells through multiple biochemical pathways, for instance, via the production of reactive oxygen species (ROS) (e.g. Klaine et al. 2008; Marambio-Jones and Hoek 2010). ROS can damage cell structures and can ultimately cause cell death (Neal 2008; Su et al. 2009). The surface-to-volume ratio increases with decreasing particle size. Thus, there is also an inverse relationship between particle size and the number of surface-oriented groups covering the particles, which is important for defining the chemical and biological properties of the nanoparticles, including generation of ROS (Nel et al. 2006; Carlson et al. 2008; Choi and Hu 2008; Neal 2008). Furthermore, the biocidal effect of most metal nanoparticles depends on their stability and resistance to agglomeration and aggregation. These properties are associated with increased release of metal ions from the larger surface area, resulting in a longer time for interaction between the nanoparticles and bacteria, and thus, a more potent antimicrobial activity (Jiang et al. 2009; Bae et al. 2010; Jin et al. 2010). However, the reactivity and biocidal properties between different metals differ greatly.
Several main mechanisms underlie the biocidal properties of silver against micro-organisms. First, Ag(0) nanoparticles attach to the cell surface, alter the physical and chemical properties of the cell membranes and the cell wall and disturb important functions such as permeability, osmoregulation, electron transport and respiration (Sondi and Salopek-Sondi 2004; Nel et al. 2009; Su et al. 2009; Marambio-Jones and Hoek 2010). Second, Ag(0) nanoparticles can cause further damage to bacterial cells by permeating the cell, where they interact with DNA, proteins and other phosphorus- and sulfur-containing cell constituents (AshaRani et al. 2009; Nel et al. 2009; Marambio-Jones and Hoek 2010). Third, Ag(0) nanoparticles release silver ions, generating an amplified biocidal effect, which is size- and dose-dependent (Lok et al. 2007; Liu et al. 2010; Marambio-Jones and Hoek 2010).
Conventional agar diffusion tests, serial dilutions and counting of colony-forming units or endpoint growth determination via turbidity measurements of the cell density are commonly used for evaluating the effects of nanoparticles on microbial biota, regardless of whether these are desired effects (e.g. against pathogens) or adverse effects on beneficial micro-organisms. By using these standard cultivation-dependent techniques for endpoint growth determination or inspection at regular time intervals, many studies have confirmed the effective biocidal activity of Ag(0)- and other metal nanoparticles against micro-organisms (e.g. Kim et al. 2007; Fernandez et al. 2008; Martinez-Castanon et al. 2008; Ruparelia et al. 2008; Egger et al. 2009; Fabrega et al. 2009; Jain et al. 2009; Travan et al. 2009; Li et al. 2010; Liu et al. 2010; Amato et al. 2011; Gottesman et al. 2011; Huang et al. 2011; Lalueza et al. 2011; Guzman et al. 2012; Oei et al. 2012).
On the other hand, researchers have demonstrated a delayed release of nanoparticle Ag(0) from processed materials (Wijnhoven et al. 2009; Benn et al. 2010), as well as successive formation of silver ions on the surface of Ag(0) nanoparticles (Lok et al. 2007; Damm and Münstedt 2008; Wijnhoven et al. 2009; Liu and Hurt 2010; Liu et al. 2010). Furthermore, nanoparticle transport, biosorption, toxicity and formation of microbial metal resistance are also subject to temporal effects and will thus largely affect microbial growth dynamics (Nies 2003; Nel et al. 2006, 2009; Harrison et al. 2007). Therefore, existing methods of acquiring growth profiles based on endpoint measurements or on analyses at discrete time points are of limited applicability for studying the entire range of dynamic effects of nanoparticle exposure on micro-organisms and the time-dependent expression of cellular response mechanisms.
This study comprises the design and application of growth tests for a reliable and time-resolved assessment of the antimicrobial properties of Ag(0) nanoparticles on microbial growth in comparison with the respective bulk material. The automated assay in 96-well microtitre plates allows simultaneous cultivation and online monitoring of microbial growth and combines high temporal resolution with the analysis of many replicate cultures.
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- Materials and methods
Nanotechnology approaches using engineered nanoparticles with biocidal properties (e.g. Ag, Zn, Cu, Ce and Ni) offer novel applications, including control of unwanted microbial colonization on diverse surfaces and prevention of biofouling, improved waste-water treatment and drinking water purification and the prophylaxis and topical treatment of infectious diseases. In contrast, the extensive use of engineered nanoparticles with antimicrobial properties and their increased release into the environment have raised major concerns due to potential (eco)toxicological effects and inappropriate testing methods. At present, there are virtually no applicable standard methodologies for evaluating the effects of exposure of microbial biota to nanoparticles, regardless of whether these are favourable effects (e.g. against pathogens) or adverse impacts in the environment.
Commonly used techniques, such as agar diffusion tests, are often inhibited by nanoparticle agglomeration or aggregation and lowered nanoparticle transport due to interactions with media components or the solidified agar matrix (Gallant-Behm et al. 2005; Dhawan et al. 2009; Jin et al. 2010; Römer et al. 2011). As a result of nanoparticle re-aggregation, these tests usually provide inconclusive results or can underestimate nanoparticle toxicity (Jiang et al. 2009; Liu et al. 2009; Bae et al. 2010; Römer et al. 2011). Conventional growth experiments in liquid media in culture bottles are highly laborious and require regular sampling and offline measurements. Therefore, such testing is frequently restricted to a small number of replicates, limited to endpoint growth determination or analysis at discrete time points. Such growth analyses may not cover details of the entire course of cultivation and are of low temporal resolution and will not recognize shifted or long-term effects. Hence, they are inadequate for tracking the finer temporal changes of microbial growth affected by nanoparticles, which may, however, convey important information for a sound evaluation of nanoparticle-mediated effects.
Factors that influence microbial growth dynamics include the delayed release of Ag(0) from processed materials and the successive generation of silver ions, cell sorption and interaction of Ag(0) nanoparticles with cellular components, the manifestation of toxic effects and the formation of cellular stress response mechanisms (Nel et al. 2006, 2009; Lok et al. 2007; Damm and Münstedt 2008; Wijnhoven et al. 2009; Benn et al. 2010; Liu and Hurt 2010; Liu et al. 2010). Expression of a diverse array of metal resistance strategies evolved by the bacteria to encounter heavy metal toxicity comprises reduction or modification of the heavy metals to less toxic species, chelation, sequestration, reduced uptake, efflux mechanisms and increased expression of the cellular repair machinery (Nies 2003; Harrison et al. 2007).
In this study, we established a 96-well-based assay that was adapted for time-resolved testing of the dynamic effects of Ag(0) nanoparticles on micro-organisms. This method allowed simultaneous cultivation and online analysis of microbial growth. The automated high-throughput assay, which combined monitoring of microbial growth rates at high temporal resolution with analysis of many replicates, was applied to C. necator test organisms to test the antimicrobial effects of the nanoparticles. Concentrations above 80 μg ml−1 Ag(0) nanoparticles revealed complete and irreversible growth inhibition, whereas Ag(0) concentrations of ≥20 μg ml−1 resulted in partial repression of bacterial growth and correlated with extended lag phases. However, reduced optical density in the plateau phase was only partly related to Ag(0) concentration and the corresponding growth inhibition (Fig. 4a). The maximum slope of the growth curves during the exponential phase was also not a measure of growth inhibition in the concentration range studied (Fig. 4b). Even more intriguing, compared with the Ag(0)-free controls, treatment with Ag(0) nanoparticles resulted in higher maximum growth rates after the extended lag phases at all concentrations tested between 20 and 60 μg ml−1 (Fig. 4b). This was similar to the effects induced by a delayed addition of Ag(0) nanoparticles, which caused a weaker inhibition with increasing time of nanoparticle treatment, indicating that the susceptibility of the micro-organisms to Ag(0) nanoparticles is growth-stage dependent.
Contrary to our expectations, in cultures that received Ag(0) nanoparticles at ≥9 h of growth, a reversible growth inhibition could be compensated for by higher growth rates until the end of the experiment (Fig. 5). These observations support the assumption that during particular growth phases micro-organisms might experience partial growth stimulation under moderate stress conditions in comparison with cultures without Ag(0) treatment. This aspect should be taken into account when treating unwanted micro-organisms, such as pathogens and food contaminants, with antimicrobial metal nanoparticles. It also suggests that for a comprehensive evaluation of nanoparticle toxicity against micro-organisms, the common use of nontemporal growth profiles or endpoint growth measurements is questionable, and full analysis will require monitoring of complex growth dynamics.
Nanoparticle-specific background signals frequently interfere with turbidity measurements in microbiological tests used for optimizing nanoparticle formulations or for risk assessment. An important prerequisite for testing metal nanoparticle suspensions with high background was the ability of the monochromator-based detection system to permit one-nm resolved measurements for the optical density. This allowed validated adjustment of the analytical settings for data acquisition and improved signal-to-noise ratios resulting in highly reproducible data, even from a large number of separate cultures in different microtitre plate wells. The high performance of the technique is associated with its ability to be easily implemented in routine lab applications. In addition to other established methods for kinetic analyses, such as isothermal microcalorimetry, it will permit a comprehensive and profound growth-dependent assessment of inhibiting agents, specifically for the elucidation of the effects by Ag(0) nanoparticles.