Size-Dependent Bacterial Growth Inhibition and Mechanism of

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Size-Dependent Bacterial Growth Inhibition and Mechanism of Antibacterial Activity of Zinc Oxide Nanoparticles Krishna R Raghupathi,† Ranjit T Koodali,†,* and Adhar C Manna‡,* †

Department of Chemistry, ‡Basic Biomedical Sciences, Sanford School of Medicine, University of South Dakota, Vermillion, South Dakota 57069, United States . ABSTRACT: The antibacterial properties of zinc oxide nanoparticles were investigated using both Gram-positive and Gram-negative microorganisms. These studies demonstrate that ZnO nanoparticles have a wide range of antibacterial activities toward various microorganisms that are commonly found in environmental settings. The antibacterial activity of the ZnO nanoparticles was inversely proportional to the size of the nanoparticles in S. aureus. Surprisingly, the antibacterial activity did not require specific UV activation using artificial lamps, rather activation was achieved under ambient lighting conditions. Northern analyses of various reactive oxygen species (ROS) specific genes and confocal microscopy suggest that the antibacterial activity of ZnO nanoparticles might involve both the production of reactive oxygen species and the accumulation of nanoparticles in the cytoplasm or on the outer membranes. Overall, the experimental results suggest that ZnO nanoparticles could be developed as antibacterial agents against a wide range of microorganisms to control and prevent the spreading and persistence of bacterial infections.

’ INTRODUCTION Inorganic metal oxides are being increasingly used for antimicrobial applications. The main advantages of using inorganic oxides when compared with organic antimicrobial agents are their stability, robustness, and long shelf life. The antimicrobial activity of nanoparticles have been studied with different pathogenic and nonpathogenic bacteria such as Staphylococcus aureus and Escherichia coli.1-6 Some of the inorganic oxides that have been tested for their antimicrobial activity are TiO2, ZnO, MgO, CaO, CuO, Al2O3, Ag2O, and CeO2.7,8,4-18 Among these, suspensions of TiO2 are effective at killing bacteria such as E. coli, S. aureus, and viruses under UV light.7,14,15 The antibacterial activity of ZnO has been studied largely with different pathogenic and nonpathogenic bacteria such as S. aureus and E. coli.5,6,8,9 ZnO nanoparticles are believed to be nontoxic, biosafe, and biocompatible and have been also used as drug carriers, cosmetics, and fillings in medical materials.19 Several reports have addressed the harmful impact of nanomaterials on living cells, but relatively low concentrations of ZnO are nontoxic to eukaryotic cells.14,20-24 We have earlier shown in a compara tive study that, among six metal oxide nanoparticles, ZnO nanoparticles significantly inhibit growth of a wide range of pathogenic bacteria under normal visible lighting conditions.6 Several studies suggest that different morphologies (particle size and shape) of ZnO have different degrees of antibacterial activities.6,13,25-27 Several studies have proposed that aqueous suspensions of ZnO produce increased levels of reactive oxygen species (ROS), mostly hydroxyl radicals, H2O2 and singlet oxygen, which contribute to the antibacterial activity of ZnO nanoparticles.15,26-32 Sawai reported that H2O2 may be an r 2011 American Chemical Society

important factor.7 Along the same lines, Zhang et al. suggested that chemical interactions between H2O2 and bacteria was the dominant mechanism for the antibacterial activity.10 The surface abrasiveness of ZnO nanoparticles was reported to produce disorganization of both cell wall and cell membrane of E. coli and thought to be responsible for high activity of ZnO nanoparticles.13 The group of Gedanken identified the formation of 3 OH and 1O2 species when a suspension of ZnO was examined by electron spin resonance (ESR) spectroscopy.30,32 Brayner et al. reported that ZnO caused damage to the membrane of E. coli, which led to accumulation of ZnO and cellular internalization.5 An alternative hypothesis suggested that the binding of ZnO nanoparticles to the bacterial surface is due to electrostatic forces that directly kill bacteria.7 Although there are numerous studies regarding the antibacterial effect of ZnO, focus has been on E. coli with relatively few reports on S. aureus. Little is known regarding interaction of nanoparticles with other bacteria, and, importantly, less is known about the mechanism underlying the antimicrobial effects. Our previous studies with two commercial ZnO nanoparticles possessing different particle sizes demonstrated the ability of these particles to reduce growth rates of several pathogenic bacteria, with the activity being size dependent.6 Specifically, the sizedependent activity was studied in the range 100 nm to 0.8 μm in S. aureus and E. coli25 and 12, 45, and 200 nm range in E. coli,13 however little is known about the activity of ZnO nanoparticles in Received: December 4, 2010 Revised: February 18, 2011 Published: March 14, 2011 4020

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Langmuir the 10-300 nm range toward S. aureus. Toward this purpose, we synthesized seven ZnO nanoparticles of varying sizes and tested their antibacterial activity using either turbidity or plate assays to understand their antibacterial activities. Our results confirm that smaller sized ZnO nanoparticles indeed have higher antibacterial activity than larger sized nanoparticles under normal lighting conditions. Furthermore, studies on different species of potential pathogenic and nonpathogenic microorganisms from various clinical isolates of bacteria demonstrate that ZnO has a wide target of antibacterial activity. Transcriptional analysis of cells treated with ZnO nanoparticles demonstrated marginal or no enhanced expression of several ROS specific genes, which suggests that ROS may not be the only factor for the antibacterial activity of ZnO nanoparticles. Finally, confocal laser scanning microscopy results suggest that treatment of bacteria with small sized ZnO nanoparticles leads to an increase in cell death, probably due to disruption of the bacterial cell wall.

’ MATERIALS AND METHODS Synthesis of ZnO Nanoparticles. Analytical grade reagents; zinc acetate dihydrate (∼98%, Acros), zinc nitrate hexahydrate (∼98%, Acros), zinc sulfate heptahydrate (99%, Acros), sodium hydroxide (extra pure, Acros), potassium hydroxide (Acros), tetramethyl ammonium hydroxide 25% in water (TMAOH) (Acros), and methanol (Acros) were used. Tryptic soy broth (TSB) (B.D.S.) and agarose (Invitrogen) were used as received without any further purification. Synthesis of ZnO nanoparticles was conducted by two methods, room temperature synthesis, and solvothermal synthesis.11,34,35 Initially, ZnO nanoparticles were prepared at room temperature and their antibacterial activity assessed by monitoring the optical density of a suspension consisting of ZnO and the bacteria. In a typical room temperature synthesis, zinc salt was dissolved in nanopure water with continuous stirring at room temperature; subsequently, a base (NaOH or KOH) was added at the rate of 5 mL per minute and hydrolysis was continued for two hours at room temperature. The precipitate was washed several times with nanopure water to remove soluble impurities, filtered through a sintered glass crucible, dried in a static air oven at 70-80 °C for 12 h, and stored in a glass vial before use. In the solvothermal method, the first step for the preparation of ZnO is analogous to the room temperature synthesis. However, after two hours of hydrolysis, the mixture was subjected to sonication for 30 min to impart uniformity and the final mixture was transferred into a Teflon-lined solvothermal bomb (autoclave). The container was placed in a muffle furnace (Sybron-Thermolyne) at a preset constant temperature of 80 °C for 24 h. After the solvothermal reaction was completed, the container was removed from the furnace and immediately cooled using water. The precipitate obtained was filtered through a sintered glass crucible and dried for 12 h in a static air oven at 80 °C. The white dried powder of ZnO was ground with a mortar and pestle and stored at room temperature in a glass vial. Characterization of ZnO Nanoparticles. The crystal structure, size, and shape of the prepared ZnO nanoparticles were investigated using nitrogen physisorption (N2), powder X-ray diffraction (XRD), scanning electron microscopy (SEM), and transmission electron microscopies (TEM). The N2 isotherms were carried out on a Quantachrome Nova 2200e series surface area analyzer at 77 K. The surface areas of ZnO nanoparticles were calculated using the Brunauer-Emmett-Teller equation

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in the relative pressure range (P/Po) of 0.05-0.30. The average particle diameter (D) of the zinc oxide particles was estimated using the equation (D = 6/S F), where S is specific surface area per unit g of the sample and F is the density of zinc oxide. This method leads to the calculation of average particles sizes and does not indicate the variability in particles and thus should be used only as a rough guide for comparison purposes only. Our average particle sizes reported are consistent with TEM studies. Powder XRD was performed on Rigaku Ultima IV diffractometer operated at 40 kV and 44 mA using a Ni filtered CuKR radiation with wavelength of 1.5408 Å in wide angle region from 24° to 75° on 2θ scale. The data were analyzed using PDXL software (Rigaku). The size and morphology of the synthesized nanoparticles were also determined by scanning electron microscope (FEI 450) and transmission electron microscope (Hitachi H-7000 and Tecnai G2). SEM images were typically recorded at 20 kV and 2
10-6 Torr. TEM images were recorded by sonicating synthesized zinc oxide nanoparticles in ethanol and carefully placing a drop of dilute ethanol containing ZnO solution on a carbon-coated copper grid and the particle sizes were determined. The soluble zinc ion concentrations in the colloidal ZnO nanoparticles suspensions were determined by atomic absorption spectrophotometer (AAS Thermo Jarrell Ash) using a hollow cathode lamp with a standard wavelength of 213.9 nm. A calibration plot was made using known zinc ion concentrations and used for calculation of the soluble zinc ion concentrations. Antibacterial Activity of ZnO Nanoparticles. The antibacterial activity of zinc oxide nanoparticles toward various microorganisms was performed using culture turbidity as a qualitative measure of cell growth and also by plating assays to compare cell viability as described.6 Various microorganisms, including Grampositive (methicillin sensitive S. aureus strains RN6390, SH1000, Newman, and UAMS-1; methicillin resistant S. aureus hospital associated, HA-MRSA, strain COL and community associated, CA-MRSA, strain MW2; Staphylococcus epidermidis; Streptococcus pyogenes N315; Enterococcus faecalis; Bacillus subtilis; and B. cereus) and Gram-negative (Escherichia coli, Proteus vulgaris, Salmonella typhimurium, Shigella flexinari, Pseudomonas alcaligenes, and Enterobacter aerogenes) strains were used to test the antibacterial activity of ZnO nanoparticles. To examine the bacterial growth rate or the bacterial growth behavior in the presence of various sized ZnO nanoparticles, different microorganisms were grown in tryptic soy broth (TSB) or LuriaBertani (LB) medium supplemented with various concentrations of ZnO colloidal suspensions. The bacterial growth curves were achieved by measuring the optical density (OD) of the cultures and plotting it against time. OD measurements were made using a Spectronic 20 instrument at 600 nm and the background (turbidity due to growth medium and ZnO suspension) was eliminated by taking blank readings. The bacteria for each experiment were freshly prepared by inoculating a miniscule amount (using a toothpick and scraping the sides of an Eppendorf tube containing the culture of frozen bacteria) into 10 mL of sterile TSB in a culture tube. The bacteria were allowed to grow overnight in an incubator maintained at 37 °C and shaken at 220 rpm. ZnO suspensions (250 mM stock suspension) for these experiments were freshly prepared by rigorously mixing the calculated amount of ZnO powder in sterile nanopure water. Overnight TSB cultures (50 μL) containing the relevant strain of bacteria were added to test tubes containing 10 mL of TSB. Appropriate concentrations of ZnO suspensions (60-420 μL) were added to the test tube and placed in a shaker at 37 °C. The 4021

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Langmuir OD600 nm of this suspension (containing the bacteria and ZnO) was periodically monitored. Cultures from selected concentrations and growth points were plated onto tryptic soy agar (TSA) or LB agar plates and incubated at 37 °C incubator to determine the presence of viable bacteria. Isolation of Total Cellular RNA and Northern Blot Hybridization. S. aureus strain SH1000 was allowed to grow to an optical density of approximately 1.2 (exponential growth phase) and various ZnO (8 nm NS, Nanoscale Materials Inc., KS; 12 ( 2 nm synthesized in this study; and bulk, Sigma-Aldrich, USA) of 0 mM and 5 mM were added to the growing cultures for one hour of incubation before harvesting the cells. Total RNA from S. aureus treated with or without ZnO was prepared using a Trizol isolation kit (Gibco BRL, Gaithersburg, MD) and a reciprocating shaker, as described.36-38 The concentration of total RNA was determined by measuring the absorbance at 260 nm. Ten micrograms each of total RNA was analyzed by Northern blotting, as described.36-38 The genes coding for sodA, sodM, trxA, trxB, ahpCF, perR, and sarA were either amplified by PCR or excised from plasmids containing desired genes with suitable restriction endonucleases. For detection of specific transcripts, gel-purified DNA probes were radiolabeled with [R-32P] dCTP by using the random-primed DNA labeling kit (Roche Diagnostics GmbH). Hybridization was performed overnight under aqueous phase conditions at 65 °C. After hybridization the blots were washed, exposed to phosphoimager screen, and scanned on a PhosphoImager (Amersham Life Sciences). Confocal Laser Scanning Microscopic Studies. Confocal microscopy studies were performed on an Olympus confocal laser scanning microscope utilizing an argon laser of 488 nm wavelength equipped with a 505 filter to monitor the emission from the ZnOstained samples at a magnification of 60
(numerical aperture was typically 1). S. aureus cultures were labeled with either fluorescein isothiocyanate (FITC), propidium iodide (PI), or both dyes.39 FITC and PI were used to determine the relative proportion of live and dead cells as PI specifically stains only dead bacteria, whereas FITC stains both the live and dead bacteria. FITC-stained ZnO was prepared in the following manner. In a typical method, 0.5 g of ZnO powder was added to 10 mL of FITC dye at 10-4 M concentration in ethanol and stirred for 10 min. The mixture was then centrifuged, washed, and dried. Staining with PI was performed by adding 50 μL of overnight grown bacterial cells to 10 mL of 5 mM ZnO TSB solution. This solution was incubated for 6 h at 37 °C. Similarly a control (not treated with ZnO) bacterial cell was grown under similar conditions. Then, 1 mL of both of these bacterial suspensions was harvested by centrifuging at 10 000 rpm for 2 min. The bacterial pellet was then stained with FITC, PI, and both PI and FITC simultaneously. The actual staining process involves the following two steps: a) staining the bacterial cells separately with 1 mL of 50 μg/mL PI in phosphate buffered saline (PBS), or with 1 mL of FITC (2.5 μg/mL) in PBS; b) dual staining with both FITC and PI. In this method, 1 mL of 50 μg/ mL PI in PBS was incubated for 10 min at 37 °C, the sample was then centrifuged and resuspended in 1 mL of FITC (2.5 μg/mL) in PBS. A few drops of the above suspensions were then placed on a glass slide, mounted with a coverslip, and analyzed under a confocal scanning microscope.

’ RESULTS AND DISCUSSION Synthesis and Characterization of ZnO Nanoparticles. A series of zinc oxide nanoparticles were initially synthesized at room temperature by employing different zinc precursors (zinc

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Table 1. Different Textural Properties of Various Sizes ZnO Particles Obtained Using Different Synthesis Conditions nanoparticle

basea

precursor

Hb methodc surface aread size (nm)e

ZnO-1

Zn(NO3)2

TMAOH 10 H.T.

90.4

12

ZnO-2 ZnO-3

Zn(NO3)2 Zn(NO3)2

TMAOH 10 H.T. KOH 5 R.T.

42.8 35.6

25 30

ZnO-4

Zn(NO3)2

NaOH 1

R.T.

12.1

88

ZnO-5

Zn(NO3)2

NaOH 5

R.T.

7.52

142

ZnO-6 ZnO-7

ZnSO4

1

R.T.

5.05

212

Zn(CH3COO)2 NaOH 1

KOH

R.T.

3.49

307

a

TMAOH refers to the base, tetramethylammonium hydroxide, (CH3)4NOH b H refers to base/Zn2þ ratios, i.e ([OH]-/[Zn2þ]) c H. T. refers to hydrothermal method and R.T. refers to room temperature d The surface areas were calculated by applying the Brunauer-Emmett-Teller (BET) equation to a relative pressure (P/Po) ranging from 0.05 to 0.3 in the adsorption isotherm. e From the equation (D = 6/S
F) where D = average particle diameter, S = specific surface area, and F = density of ZnO (5.6 g/mL).The chemicals and the conditions used were similar for ZnO-1 and ZnO-2, however the rate of addition of Zn(NO3)2 was 2 mL/min and 5 mL/min respectively.

acetate dehydrate, zinc nitrate hexahydrate, or zinc sulfate heptahydrate) and different bases (sodium hydroxide or potassium hydroxide) to standardize optimal conditions for the preparation of ZnO nanoparticles with various particle sizes. Table 1 gives a summary of the results obtained from the synthesis conducted at room temperature. From Table 1, it can be observed that the ZnO particle sizes using zinc acetate and zinc sulfate were relatively higher than those obtained using zinc nitrate as a precursor. Though the reason for this effect is not exactly known, we can hypothesize that the solubility of the salts in the aqueous solutions may have played a role. The solubility of zinc nitrate hexahydrate is 200 g/100 mL, which is higher than the solubility of either zinc acetate or zinc sulfate (43 and 54 g/ 100 mL, respectively). We also observed that ratios ([OH]/[Zn2þ]) of 1, 5, or 10 were optimal for obtaining relatively large yields of ZnO and subsequent preparative methods used these base/Zn2þ ratios. As Table 1 indicates, a wide range of particle sizes were obtained by employing the room temperature method allowing us the ability to examine antibacterial activity in this broad range. Attempts to prepare smaller ZnO nanoparticles (less than 30 nm) by varying time, temperature of synthesis, and solvent and so forth were not successful and hence we used solvothermal synthesis. The room temperature synthesis was modified to the solvothermal method11,34,35 using the optimal precursor and the base compositions as this method has been described to yield ZnO nanoparticles with relatively smaller particle sizes.34 The solvothermal synthesis method led to the synthesis of ZnO nanoparticles with diameters of 12 ( 2 nm and 25 ( 3.6 nm nanometers as indicated in Table 1. Thus, a combination of room temperature and solvothermal synthesis led to the preparation of different sized ZnO nanoparticles with diameters of 12 ( 2, 25 ( 3.6, 30 ( 4.0, 88 ( 8, 142 ( 12, 212 ( 5, and 307 ( 6 nm respectively. The ZnO nanoparticles were characterized by powder XRD, N2 physisorption, and electron microscopy (SEM and TEM). The powder XRD data indicate high crystallinity and a typical XRD pattern of a 12 ( 2 nm diameter synthesized ZnO nanoparticles is shown in part A of Figure 1. The sample show peaks at the positions of 2θ = 31.63°, 34.50°, 36.25°, 47.50°, 56.50°, 62.80°, 66.35°, 67.92°, and 68.91°, which are in good agreement with the assigned standard 4022

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Figure 1. Physical characterization of ZnO nanoparticles (average particle diameter ∼12 ( 2 nm) synthesized using zinc nitrate and base tetramethylammonium hydroxide (TMAOH) by the solvothermal method. A: powder XRD pattern; B: nitrogen isotherm analysis, C: transmission electron microscope (TEM) image; D: selected area electron diffraction pattern, and E: scanning electron microscopy image.

wurtzite-type ZnO structure (JCPDS 36-1451). The formation of amorphous species cannot be detected by powder XRD studies and it is possible that our room temperature and hydrothermal synthesis led to the formation of amorphous Zn(OH)2. To verify this, we calcined three representative materials at 250 and 400 °C, respectively. The weight losses for ZnO-1, ZnO-2, and ZnO-3 after heating to 250 °C were determined to be 0.13, 0.14, and 0.096%. The weight losses for ZnO-1, ZnO-2, and ZnO-3 after heating to 400 °C were found to be 0.21, 0.26, and 0.23%. This indicates the absence of any Zn(OH)2. Thus, the powder XRD results demonstrate successful formation of crystalline ZnO nanoparticles. Nitrogen physisorption studies indicate that the materials are predominantly mesoporous in nature and a relatively large surface area (∼90 m2/g) can be obtained by the hydrothermal synthesis method. The nitrogen isotherm of a hydrothermally synthesized ZnO material is shown in part B of Figure 1. They exhibit type IV isotherms (H3 type of hysteresis loop) typical of mesoporous materials. The appearance of hysteresis is a reflection of the porosity and a result of pore filling and emptying taking place separately. The initial part of the type IV isotherm is due to monolayer adsorption at low relative pressures. As the relative pressure increases, multilayer adsorption occurs and is followed by capillary condensation. After all of the pores are filled, the adsorption isotherm usually levels. However, as seen in part B of Figure 1, these materials do not exhibit limiting adsorption at high saturation pressures (P/P0), suggesting that the materials are comprised of loose aggregates of particles forming slit-like pores. Part C of Figure 1 shows the morphology of ZnO using transmission electron microscopy (TEM). The 2D lattice fringes indicate the high crystallinity of the ZnO nanoparticles, and the distance between two fringes is 0.25 nm consistent with (101) reflection of ZnO (JCPDS 36-1451). The ZnO particles are

fairly agglomerated and the average particle diameter calculated from the XRD results and the particle sizes from TEM studies are consistent. In addition, the selected area electron diffraction (SAED) shown in part D of Figure 1 is an indication of the high crystallinity of the ZnO nanoparticle. Part E of Figure 1 shows the SEM images, which indicate that the primary ZnO nanoparticles aggregate to form larger secondary particles. Size-Dependent Antibacterial Activity of the Synthesized ZnO Nanoparticles toward Staphylococcus aureus. In the previous studies, we tested six metal oxides nanoparticles (MgO, TiO2, CuO, CaO, CeO2, and ZnO). Among them, ZnO nanoparticles showed significant growth inhibition in a size-dependent manner under normal ambient lighting conditions.6 We also reported that the smaller commercial ZnO nanoparticles of 8 nm in diameter have significant growth inhibition toward different microorganisms including S. aureus, E. coli, and B. subtilis.6 In this work, we present a detailed and systematic study to understand the effect of particle sizes on antibacterial activity. To determine the antibacterial activity of the synthesized ZnO nanoparticles, we performed both liquid broth and plate based growth studies in methicillin sensitive S. aureus (MSSA) as described earlier.6 Antibacterial activity of the ZnO nanoparticles was dose dependent. The minimum inhibitory concentration (MIC) was generally observed to be in the range of 4 to 7 mM (depending on the particular bacterial strain) and these concentrations were most commonly used. As shown in part A of Figure 2, the results demonstrate that size-dependent bacterial growth inhibition of S. aureus exists in the presence of 6 mM of different sizes of ZnO nanoparticles. In addition to further verify, the viable S. aureus cells were determined by plating cultures from the growing cells in the presence of 6 mM concentration of different sized ZnO nanoparticles. At a given point of growth, it is important to note that viable cells recovered decreased significantly with decreasing 4023

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Figure 2. Effect of different sizes ZnO nanoparticles on the growth of methicillin sensitive S. aureus strain. A. Growth analysis curves measured by monitoring the optical density (OD) at 600 nm, and B. the viable S. aureus 6390 recovered from TSA plates after 24 h of incubation at 37 °C. Cultures were grown in the presence of 6 mM of various sizes ZnO nanoparticles and after 6 h cultures were diluted to 10-6 and placed on TSA plates in triplicates. Colonies were counted and the percentage of growth inhibition was calculated and plotted against particle sizes. The experiments were repeated at least three independent times and the average data was presented in the figures.

particle size of ZnO nanoparticles (part B of Figure 2). These results are consistent with an earlier finding that the antibacterial activity of ZnO varied with particle sizes in the range of 100 nm to 0.8 μm for S. aureus and E. coli25 and 200 to 12 nm for E. coli.13 Furthermore, in the previous study, the influence of different sizes ZnO nanoparticles was not demonstrated using S. aureus. The antibacterial activity exhibited by ZnO nanoparticles could also be due to the presence of soluble Zn2þ formed when ZnO is suspended in water or due to changes in pH since the bacterial activity is sensitive to both of these factors. The solubility of metal oxide nanoparticles increases as their particle size decreases, and the enhanced activity of the smaller sized ZnO nanoparticles could also be due to the formation of dissolved Zn2þ ions. Thus, the soluble Zn2þ concentrations of different ZnO samples in aqueous medium were determined by AAS (Atomic absorption spectrophotometry) experiments. Zn2þ ion concentration in synthesized ZnO nanoparticles (∼12 nm) was measured and compared with zinc nitrate hexahydrate by preparing colloidal suspension and solution respectively at room temperature. The dissolved Zn2þ ions from the suspension was separated by high speed centrifugation and the concentration and antibacterial activity was monitored in atomic absorption spectrophotometer and growth inhibition assays, respectively. The concentration of Zn2þ ions was determined to be 354-fold higher in the zinc nitrate solution compared to a solution of 1 mM ZnO nanoparticles. Growth inhibition was not observed (100 nm), no significant growth inhibition in the methicillin sensitive S. aureus RN 6390 was observed at the 6 mM dose of ZnO. Surprisingly, the bacteria were not completely killed, that is the ZnO functioned only as a bacteriostatic agent. However, as shown in Figure 3, when the smallest particle size of ZnO was used (∼ 12 nm), ZnO not only limited the growth of S. auereus RN 6390, but also effectively killed them. Antibacterial Effects on Other Staphylococci Strains and Microorganisms. There have been extensive studies to evaluate the antibacterial effect of ZnO and other oxide nanoparticles in vivo using mostly E. coli and occasionally S. aureus. In the present study, our interest was to determine if ZnO nanoparticles could be employed to inhibit the growth or kill several other diverse bacterial strains. Some of the pathogens that were examined in this study are different isolates of methicillin sensitive (laboratory strains RN6390 and SH1000, highly virulent clinical strain Newman, and osteomyelitis isolate strain UAMS-1), methicillin resistant (first isolated hospital-associated MRSA strain Cowan and community associated MRSA strain, MW2) S. aureus, a high biofilm producing strain S. epidermidis 1487, Streptococcus pyogenes N315, Enterococcus faecalis, Bacillus subtilis, B. cereus, Proteus vulgaris, E. coli, Salmonella typhimurium, Pseudomonas alcaligenes, Enterobacter aerogenes, and the dysentery causing Shigella flexneri. Figure 4 represents the growth inhibition assays for several bacterial pathogens. We demonstrate that the concentration range of 4-7 mM colloidal suspension of ZnO nanoparticles (particle size ∼12 nm) could inhibit more than 95% of growth for most of the microorganisms, except Salmonella typhimurium whose growth was inhibited by about 50% with 5 and 10 mM of ZnO nanoparticles under our experimental conditions. The patterns of growth inhibition of other microorganisms were similar and 95% growth inhibition was achieved with a concentration of 5 mM colloidal suspension of ZnO nanoparticles (data not shown). The viable cell counts 4024

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Figure 3. Plate assay of S. aureus 6390 using control (left panel) and ZnO nanoparticles (∼12 nm sized, right panel). The experiments were repeated at least three independent times and the average data was presented in the figures.

Figure 4. Growth curves of various microorganisms in the presence of different concentrations of ZnO nanoparticles with average particle diameter of 12 nm. Cultures were set up and grown under the same conditions as described in Figure 2. The different strains of microorganisms are used in various panels are: A) S. aureus strain MW2 (CA-MRSA), B) S. aureus strain Newman (MSSA), C) S. aureus strain Cowan (HA-MRSA), D) Proteus vulgaris, E) Salmonella typhimurium, and F) Shigella flexinari (left) and Bacillus cereus (right). Growth assays of various bacterial strains were done using tryptic soy broth (TSB) in panels A-C; whereas Luria-Bertani (LB) media was used in panels D-F in the presence of different concentrations of ZnO nanoparticles (∼12 nm particle diameter). The percentage of growth inhibition at 6 h of growth is shown in panels D-F. The experiments were repeated at least three independent times and average data was presented in the figures.

from the selected points of growth of the microorganisms demonstrated that the number of recovered bacteria was significantly less (nearly 95%) compared to the untreated control. Thus, these studies demonstrate that the synthesized nanoparticles of ZnO have significant antibacterial effects on a wide range of microorganisms under normal lighting conditions. However, much of the differential antibacterial activity of ZnO nanoparticles on various microorganisms will depend on cell wall integrity or membrane structures of the respective bacteria as the outer membrane structure of Gram-negative bacteria is remarkably different from that of Gram-positive bacteria. It is also well-

known that different strains within a species vary significantly in term of infectivity, and tolerance to various agents including antibiotics. Mechanism of the Antibacterial Activity of ZnO Nanoparticles. Several studies have suggested, two possible mechanisms could be involved in the interaction between nanoparticles and bacteria - (1) the production of increased levels of reactive oxygen species ROS), mostly hydroxyl radicals and singlet oxygen15,26-32 and (2) deposition of the nanoparticles on the surface of bacteria or accumulation of nanoparticles either in the cytoplasm or in the periplasmic region causing disruption of 4025

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Figure 5. Transcriptional analysis of various reactive oxygen species (ROS) specific target genes under different growth conditions in the wild type S. aureus SH1000 (MSSA) and MW2 (CA-MRSA) strains. The strains were grown to the exponential phase of growth (OD600 nm of 1.2) and treated with 5 mM of different ZnO particles. After one hour of treatment, total cellular RNA was isolated and subjected to Northern analysis with trxA, trxB, sodA, sodM, katA, ahpCF, and perR gene probes. The different ZnO particles employed are NS (8 nm commercial ZnO nanoparticles, Nanoscale Materials Inc., KS, USA), Syn (12 nm ZnO nanoparticles synthesized for this study), and bulk (ZnO powder, Sigma-Aldrich, USA). The control (Cont) strains were grown under similar condition without any ZnO particles added. The region of 23S and 16S rRNA of the ethidium bromide-stained gel used for blotting is also shown as a loading control.

cellular function and/or disruption and disorganization of membranes.5,10 It has been suggested that ZnO nanoparticles are able to slow down E. coli growth due to disorganization of E. coli membranes, which increases membrane permeability leading to accumulation of nanoparticles in the bacterial membrane and cytoplasmic regions of the cells.5 A different protective mechanism of ZnO has been suggested in that ZnO may protect intestinal cells from E. coli infection by inhibiting the adhesion and internalization of bacteria by preventing the increase of tight junction permeability and modulating cytokine gene expression.22 We believe that both production of the ROS and accumulation or deposition of ZnO nanoparticles within the cytoplasm or on the surface of S. aureus leads to either inhibition of bacterial growth or killing of S. aureus cells. Irradiation of ZnO nanoparticles by UV light (flux of photons from the lamp was ∼6
10-6 Einstein’s/s in the 300-400 nm wavelength range) provided by Xe arc lamp indicated a further reduction in the viable-cell count. Very few colonies were detected in plates for shorter exposure (15 min) of the culture containing 5 mM of ZnO nanoparticles, whereas none were detected for a longer exposure (30 min) in comparison with bacteria that were irradiated in the absence of ZnO. The enhanced antibacterial activity in this instance is due to the increased production of the reactive oxygen species (ROS) in the presence of ZnO and UV light. Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion (O2-), hydroxyl radicals (OH 3 ), and organic hydroperoxides (OHPs) are toxic to the cells as they damage cellular constituents such as DNA, lipids, and proteins.40 The role of ROS in antibacterial studies has been the subject of intense debate, and a general consensus seems to be elusive.41-43 Interestingly, S. aureus has developed efficient pathways to defend against oxidative stresses by increasing expression of oxidative stress-responsive gene products such as superoxide dismutase (encoded by the sodA and sodM genes and converts O2- to H2O2), catalase (the katA gene product converts H2O2 to H2O and O2), thioredoxin

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reductase (encoded by trxB; maintains thioredoxin in reduced form to protect cell against toxic oxygen species), thioredoxin (encoded by trxA), alkyl hydroperoxide reductase sub C and F (ahpCF, having activity against organic peroxides, peroxynitrate and H2O2), and endopeptidase (clpC) in combating oxidative stress.38,39 We performed transcriptional analysis of several genes those are directly involved in the ROS neutralization in the cells as shown in Figure 5. Analysis for both the MSSA and MRSA strains clearly suggests that a marginal or no change of transcription of the analyzed genes with three different ZnO, a commercial 8 nm, synthesized 12 nm and bulk. The catalase (KatA) involved in neutralization of H2O2 was not affected significantly suggesting different mode of antibacterial activity of ZnO in S. aureus. Two transcriptional regulatory systems, perR (Figure 5) and sarA (data not shown), which is involved in regulation of these ROS specific genes, have no significant transcriptional effect with the treatment of ZnO. Taken together these results suggest that the production of ROS by ZnO nanoparticles or powder may not be high enough to induce the expression of the ROS specific genes in S. aureus, although, the antibacterial activity of these nanoparticles has been investigated in both the earlier and the present studies.6 We have performed growth analysis in the absence or the presence of 2.5 mM ZnO nanoparticles using both single and double mutants of the sodA and sodM genes of S. aureus. Only 20% growth inhibition was observed in the double mutant compared to the wild type or the single sodA and sodM mutant strains grown in the presence of 2.5 mM ZnO nanoparticles (data not shown). Analysis of the sod gene mutants of S. aureus suggest that ZnO nanoparticles may be involved in production of oxidative stress responses, but more analysis, in particular using catalase mutant of S. aureus, will be performed in the future to study the contribution of ROS. A further experiment was carried out to understand the role of hydrogen peroxide or any reactive oxygen species in the antibacterial activity of ZnO. For this, we prepared a uniform colloidal suspension of 5 mM ZnO nanoparticles and high speed centrifugation for 1 h was carried out to sediment ZnO nanoparticles. The supernatant was collected and growth inhibition assays with wild type S. aureus strain were performed. Growth inhibition was not observed with the supernatant solution, whereas more than 95% growth inhibition was observed in the presence of 2 mM commercial H2O2 against the wild type S. aureus strain. Our attempts to detect H2O2 were not successful under our experimental conditions. We plan to further investigate this by optimizing the quantitating ROS and using different oxidative stress responsive gene mutation strains of S. aureus. To further probe the effect of ZnO nanoparticles on the viability of S. aureus strain, we performed confocal laser scanning microscopy (CLSM) in the presence of fluorescein isothiocyanate (FITC) and propidium iodide (PI).39 It should be noted that PI can only stain the cells in which the cell membrane is disrupted since it intercalates into the double-stranded nucleic acids. However, FITC can stain both the live and dead cells because it can penetrate the cell membrane. Nanoparticles prepared in this study have high surface areas and small particle sizes and thus cause the bacteria to coagulate in nanoparticle suspensions making it challenging to observe individual bacterium. Figure 6 represents dual-stained cells of S. aureus treated with and without ZnO nanoparticles. Results from the dual staining suggest that the ZnO nanoparticles treated cells are dead compared to untreated S. aureus cells. The majority of the 4026

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Figure 6. Confocal micrographs of S. aureus treated (A) without and (B) with 5 mM of ZnO nanoparticles (average diameter size 12 nm). Both S. aureus cultures were stained with fluorescein isothiocyanate (FITC) and propidium iodine (PI) dyes. Green fluorescence is characteristic of the live cells, whereas red fluorescence is due to dead cells. ZnO nanoparticles treated S. aureus cells are found to be mostly dead due to nonpermeable PI dye. The experiments were repeated at least three independent times.

untreated cells showed green fluorescence due to the viable or live cells indicating intact cell wall structure, whereas only a small percentage of the untreated cells showed red fluorescence indicating dead cells with nonpermeable cell wall or membrane structure. In contrast, almost 100% of 5 mM ZnO nanoparticles treated cells showed red fluorescence indicating dead cells. Taken together, these results suggest that the treatment of S. aureus strain with ZnO nanoparticles leads to cell death and/or bacteriostatic effect. However, much of the function, specificity, toxicity, and efficacy of nanoparticles remain unknown and these topics will be the subject of our future research. The focus of this study was to examine the particle size effect in S. aureus and to determine if synthesized ZnO nanoparticles have antibacterial activity toward multiple microorganisms. The results presented demonstrate that we are able to synthesize high-quality ZnO nanoparticles of varying sizes using simple room temperature or solvothermal methods. The antibacterial activity of ZnO nanoparticles is dependent on the size of the nanoparticles and is mainly due to the particulate ZnO as the release of free Zn2þ ions from ZnO colloidal solution is minimal under our experimental conditions. These studies also demonstrate that ZnO nanoparticles have wide range of antibacterial effects on various microorganisms, including both Gram-positive and Gram-negative bacteria under normal lighting conditions. Interestingly, there is no difference in the antibacterial activity against methicillin sensitive and methicillin resistant S. aureus strains. Additional activation of ZnO nanoparticles in the presence of S. aureus cells with UV light leads to enhanced antibacterial activity. The antibacterial activity of ZnO nanoparticles in S. aureus may involve the production of ROS and the deposition on the surface or accumulation in the cytoplasm of the cells. It would be interesting to determine if any derivates of ZnO nanoparticles with chemical groups or bioagents are more effective at eliminating various microorganisms. Several reports have suggested that modification of nanoparticle surfaces can efficiently target and kill both Gram-positive and Gram-negative bacteria.44-46 Therefore, in the future, ZnO nanoparticle-

containing formulations may be utilized as antibacterial agents in ointments, lotions, mouthwashes, and surface coatings on various substrates to prevent microorganisms from attaching, colonizing, spreading, and forming biofilms in indwelling medical devices.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ1 605 677-6336, fax: þ1 605 677-6381, e-mail: [email protected]. (A.C.M.); e-mail: [email protected]. (K.R.R.).

’ ACKNOWLEDGMENT We would like to thank Dr. S. P. Ahrenkiel, Mr. S. Mishra of South Dakota School of Mines and Technology, and Dr. Cuikun Lin, University of South Dakota, for help with the TEM studies. Thanks are also due to Mr. T. S. Remund and Ms. F. Day from the University of South Dakota for help with SEM and CLSM studies. We thank Dr. Victor Huber for critical reading and comments on the manuscript. This work was supported by the 2010-initiative start-up fund awarded to A.C.M, and R.T.K. acknowledges support from Center for the Research and Development of Light-Activated Materials (CRDLM), NSF-CHE0722632, NSF-EPS-0903804, NSF- CHE-0840507, and DOEDE-EE0000270. ’ REFERENCES (1) Sunada, K.; Kikuchi, Y.; Hashimoto, K.; Fujishima Environ. Sci. Technol. 1998, 32, 726–728. (2) Sunada, K.; Watanabe, T.; Hashimoto, K. J. Photochem. Photobiol., A 2003, 156, 227–233. (3) Rincon, A. G.; Pulgarin, C. Appl. Catal., B 2004, 49, 99–112. (4) Liu, H.-L.; Yang, T. C.-K. Process Biochem. 2003, 39, 475–481. (5) Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fievet, F. Nano Lett. 2006, 6, 866–870. 4027

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