Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Effects of silver nanoparticles on microbial growth dynamics V.J. Schacht1,3, L.V. Neumann1, S.K. Sandhi1, L. Chen2, T. Henning2, P.J. Klar2, K. Theophel1, S. Schnell1 and M. Bunge1 1 Institute of Applied Microbiology, Justus Liebig University of Giessen, Giessen, Germany 2 Institute of Experimental Physics I, Justus Liebig University of Giessen, Giessen, Germany 3 The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 39 Kessels Road, Coopers Plains, QLD 4108, Australia
Keywords Ag, antifouling, antimicrobial nanoparticles, biocidal nanoparticles, engineered metal nanoparticles, growth inhibition, growth kinetics, silver, surface. Correspondence Michael Bunge, Institute of Applied Microbiology, Research Center for BioSystems, Land Use, and Nutrition (IFZ), Justus Liebig University of Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany. E-mail:
[email protected] 2012/0935: received 22 May 2012, revised 2 August 2012 and accepted 22 August 2012 doi:10.1111/jam.12000
Abstract Aims: Engineered metal nanoparticles are increasingly used in consumer products, in part as additives that exhibit advantageous antimicrobial properties. Conventional nanoparticle susceptibility testing is based largely on determination of nontemporal growth profiles such as measurements of inhibition zones in common agar diffusion tests, counting of colony-forming units, or endpoint or regular-interval growth determination via optical density measurements. For better evaluation of the dynamic effects from exposure to nanoparticles, a cultivation-based assay was established in a 96-well format and adapted for time-resolved testing of the effects of nanoparticles on microorganisms. Methods and Results: The modified assay allowed simultaneous cultivation and on-line analysis of microbial growth inhibition. The automated highthroughput assay combined continuous monitoring of microbial growth with the analysis of many replicates and was applied to Cupriavidus necator H16 test organisms to study the antimicrobial effects of spherical silver [Ag(0)] nanoparticles (primary particle size distribution D90 < 15 nm). Ag(0) concentrations above 80 lg ml 1 resulted in complete and irreversible inhibition of microbial growth, whereas extended lag phases and partial growth inhibition were observed at Ag(0) concentrations between 20 and 80 lg ml 1. Addition of Ag(0) nanoparticles at different growth stages led to either complete inhibition (addition of 40 lg ml 1 Ag(0) from 0 h to 6 h) or resulted in full recovery (40 lg ml 1 Ag(0) addition 9 h). Conclusions: Contrary to the expected results, our data indicate growth stimulation of C. necator at certain Ag(0) nanoparticle concentrations, as well as varying susceptibility to nanoparticles at different growth stages. Significance and Impact of the Study: These results underscore the need for time-resolved analyses of microbial growth inhibition by Ag(0) nanoparticles. Due to the versatility of the technique, the assay will likely complement existing microbiological methods for cultivation and diagnostics of microbes, in addition to tests of other antimicrobial nanoparticles.
Introduction 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
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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 selfdisinfectant 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 26
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 Mu¨nstedt 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
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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. Materials and methods Micro-organisms and culture conditions for growth Cupriavidus necator H16 (DSM 428, syn. Ralstonia eutropha) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ) and was grown according to the instructions by DSMZ, either in nutrient broth or on nutrient agar (DSMZ medium number 1) or in DSMZ medium number 81 (mineral medium for chemilithotrophic growth, H-3; www.dsmz.de). Culture bottles and Erlenmeyer flasks containing the media were autoclaved for 30 min at 122°C and subsequently stored at 4°C. All manipulations, including addition of supplementary media ingredients and inoculations of precultures (16 h), were performed by using sterile microbiological techniques. For cultures in Erlenmeyer flasks (25°C, agitation at 120 rev min 1), 1-ml samples were withdrawn at predetermined time intervals, and samples were used for routine determination of the optical density (OD660nm), total cell numbers over the incubation time and colony-forming units on growth plates using 15% (w/v) agar. Typically, two sampling bottles remained uninoculated and served as sterile controls. Frozen cultures for preservation were prepared by mixing equal amounts of pregrown cultures and 20% sterile glycerol (Carl Roth, Karlsruhe, Germany), and 2-ml aliquots were stored at 80°C. Cultivation and growth analysis of C. necator in microtitre plates Cultivation of C. necator H16 in microtitre plates was performed using sterile 96-well suspension culture plates (polystyrene microplates, flat bottom, art. 655161; Greiner bio-one, Frickenhausen, Germany) and closed with sterile standard-profile lids without condensation rings (polystyrene lids, art. 656161). Precultures (40 ml, 16 h at 25°C, agitation at 100 rev min 1) grown in 100-ml Erlenmeyer flasks were harvested, resuspended in fresh medium (10% v/v) and mixed with Ag(0) nanoparticles (AgPure W10, primary size distribution D90 < 15 nm,
Ag(0) nanoparticle effects on microbial growth dynamics
ras materials, Regensburg, Germany) which were added from 1 mg ml 1 or 5 mg ml 1 Ag(0) stock solutions, resulting in a final Ag(0) concentration of 10–100 lg ml 1. Aliquots of 250 ll of the desired master suspension (Ag(0) concentrations 0 to 100 lg ml 1) were transferred into the appropriate wells using multichannel pipettes. The stability of nanoparticle suspensions in medium was monitored by light and electron microscopy (data not shown), as well as by nanoparticle tracking analysis (NTA). Spectrophotometer wavelength scans (450–1000 nm) for Ag(0) nanoparticles were carried out at a concentration of 1 mg ml 1 Ag (0) (AgPure W10). Cell cultivation and growth analysis were performed using a Tecan infinite M200 multimode microplate reader equipped with monochromator optics. Microplates were incubated at 25°C, under orbital shaking conditions of 3-mm shaking amplitude and 15-s shaking cycles, and conditioned for 30 min for temperature equilibration before measurements were started. Measurement was performed each 15 min using the multiple-reads-per-well mode (filled-circle alignment, nine reads per well, border 1000 lm). In general, eight replicate cultures were analysed for growth at each Ag(0) concentration, along with another eight replicates per Ag(0) concentration as sterile media controls. The latter background readings were also measured at each sampling time, and all readings were normalized appropriately. Growth analysis of C. necator in the presence of Ag(0) nanoparticles was performed along with controls without Ag(0) to obtain reference growth curves. The effects of bulk Ag(0) (Ag(0) flakes, purity 999%, particle size 10 lm, Sigma-Aldrich) were determined by culturing cells under identical growth conditions to the nanoparticles. The Ag(0) flakes were diluted to 10 and 50 lg ml 1 and compared with Ag(0)-nanoparticle-mediated growth inhibition. Characterization of Ag(0) nanoparticles by nanoparticle tracking analysis The particle size distribution of suspended nanoparticles was measured for up to 72 h using nanoparticle tracking analysis (NTA). NTA was performed on a NanoSight LM14 device (NanoSight Ltd, Amesbury, UK) equipped with a 532-nm laser (50 mW) and NTA software (ver. 22) for capture and analysis of data. Dilutions of 1 : 100 were prepared in three replicates using autoclaved (30 min at 122°C) and sterile-filtered deionized water (sterile 02-lm pore size membrane filters, cellulose acetate, Whatman; Pure Lab Plus, ELGA LabWater). One ml was injected into the sample chamber using sterile syringes and sterile-filtered deionized H2O served as a particle-free control. Between analyses, the sample
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Data analysis The maximum specific growth rate, the duration of lag phase and the maximum final OD values were determined during 85 h of growth in the presence or absence of Ag(0) nanoparticles. For calculations, the normalized mean OD660nm values from cultures with 0, 20, 40 and 60 lg ml 1 Ag(0) nanoparticles were used at each of the 340 time points, respectively. The maximum specific growth rate was determined for each interval during the exponential growth phase, considering OD660nm values between 02 and 075 (OD660nm = 02 to 06 for 60 lg ml 1) and according to l = (lnx1 lnx2)/t1 t2, where, l is the growth rate, x1 is the OD660nm at time t1 and x2 is the OD660nm at time t2 (h). Average growth rates for larger intervals (1 h) were calculated from the data for growth rates at 15-min intervals. Maximum final OD660nm values were determined from the mean values (eight replicates each) between 81 h and the end of the cultivation, whereas the duration of the lag phase was read when the OD660nm had reached 02. Results Experimental design For development of an automated method for simultaneous cell culturing and monitoring of microbial growth in the presence of Ag(0) nanoparticles, a multifunctional monochromator-based instrument was used, providing continuous absorbance measurements in the range of 230 –1000 nm (1-nm increment). In addition to the simultaneous analysis of a large number of replicates, it offers the possibility of parallel cultivation and growth analysis in a microplate format with a high temporal resolution. Furthermore, the monochromator-based instrument enables adjustment of the optical measurement settings in the presence of dispersed metal nanoparticles. Metal nanoparticles frequently exhibit strong background signals (Fig. 1, insert), which impair absorbance measurements due to nanoparticle-specific and concentrationdependent properties (e.g. type of metal/metal oxide and size distribution). The absorption of Ag(0) nanoparticle suspensions in the presence or absence of test organisms was determined at wavelengths from 450 to 1000 nm. Absorbance scans of 1 mg ml 1 Ag(0) nanoparticles in medium in the 28
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chamber was cleaned by rinsing with sterile-filtered deionized water, and then, fresh water samples were analysed to exclude cross-contamination. All measurements were performed at 25°C for 90 s, and sample videos were taken at a speed of 30 frames per second.
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Figure 1 Comparison of absorption spectra from samples with (i) 1 mg ml 1 Ag(0) nanoparticles (AgNPs) in medium and in the presence of cell suspensions of Cupriavidus necator H16, (ii) 1 mg ml 1 Ag(0) nanoparticles in sterile medium controls and (iii) test cell suspensions without Ag(0) nanoparticles. The monochromator-based detection permitted adjustment of the optical measurement settings to obtain optimal absorbance signal-to-noise ratios for different types of nanoparticles. Absorbance scans show mean values of eight replicates at 1-nm incremental wavelengths from 450 to 1000 nm. Error bars indicating standard deviations are presented only for selected wavelengths. Insert: background signals of metal nanoparticle suspensions: example of a 96-well microplate with Ag(0) nanoparticles at 0, 50 and 100 lg ml 1. ( ) Cells + Growth Medium + 1 mg ml 1 AgNPs; ( ) Growth Medium + 1 mg ml 1 AgNPs; ( ) Cells + Growth Medium w/o AgNPs.
presence of test cell suspensions (C. necator H16) and of 1 mg ml 1 Ag(0) nanoparticles in sterile medium controls showed decreasing values at wavelengths of > 500 nm and reached an OD < 1 at 650 nm and of 625 nm, respectively (Fig. 1). Cell suspensions with the same cell density but without Ag(0) nanoparticles served as controls and revealed a decreasing trend, from OD = 101 (450 nm) to OD = 057 (1000 nm) (Fig. 1). Based on the high variability of OD values in samples with Ag(0) nanoparticles at
