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Antibacterial activities of silver nanoparticles and antibiotic-adsorbed silver nanoparticles against biorecycling microbes Chandni Khurana,a Anjana K. Vala,b Nidhi Andhariya,a O. P. Pandeya and Bhupendra Chudasama*a Silver nanoparticles have a huge share in nanotechnology based products used in clinical and hygiene products. Silver nanoparticles leaching from these medical and domestic products will eventually enter terrestrial ecosystems and will interact with the microbes present in the land and water. These interactions could be a threat to biorecycling microbes present in the Earth’s crust. The antimicrobial action towards biorecycling microbes by leached silver nanoparticles from medical waste could be many times greater compared to that of silver nanoparticles leached from other domestic products, since medical products may contain traditional antibiotics along with silver nanoparticles. In the present article, we have evaluated the antimicrobial activities of as-synthesized silver nanoparticles, antibiotics – tetracycline and kanamycin, and antibiotic-adsorbed silver nanoparticles. The antimicrobial action of silver nanoparticles with adsorbed antibiotics is 33–100% more profound against the biorecycling

Received 30th April 2014 Accepted 21st May 2014

microbes B. subtilis and Pseudomonas compared to the antibacterial action of silver nanoparticles of the same concentration. This study indicates that there is an immediate and urgent need for well-defined

DOI: 10.1039/c4em00248b

protocols for environmental exposure to silver nanoparticles, as the use of silver nanoparticles in

rsc.li/process-impacts

nanotechnology based products is poorly restricted.

Environmental impact Due to the antibacterial properties of silver nanoparticles (AgNPs), their production and applications have increased. AgNPs can be used in various areas including clinical and hygiene products. During the production, use, and disposal of these products, nanoparticles can be released into the environment. Their exposure to the environment may have a harmful impact on biorecycling microbes. To study their environmental risks, a comprehensive understanding of the source, distribution and toxicity of AgNPs is needed. This article studies the effect of AgNPs and antibiotic-adsorbed AgNPs on environmentally friendly bacterial strains.

Introduction In a recent project report on “Emerging Nanotechnologies” by Woodrow Wilson International Center for Scholars, it is reported that 1600+ nanomaterial-incorporated consumer products are available on the market for commercial use.1 The market share of nanotechnology based products has grown at an impressive rate of 379% in the last ten years. In the United States alone, the nanomaterial business will be a trillion dollar industry by 2015.2 Antimicrobial silver nanoparticles (AgNPs) are one of the most widely used engineered nanomaterials in nanotechnology based products.3 Thanks to their biocidal activity, AgNPs are routinely incorporated into medical supplements, catheters, wound dressings and implants to

a

School of Physics & Materials Science, Thapar University, Patiala, 147 004, India. E-mail: [email protected]; Fax: +91-175-2393020; Tel: +91-175-2393116

b

Department of Physics, M.K. Bhavnagar University, Bhavnagar, 364 022, India

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inhibit pathogen growth. AgNPs are added to cosmetics as an antiseptic and used in medical textiles to eliminate microbes in the clinical environment.3–6 They are also used for water purication, biosensors, bone prostheses, drug and gene delivery, bioimaging devices, etc.7–10 The majority of AgNPs released from consumer products are expected to enter terrestrial ecosystems through the land application of biosolids.11,12 While environmental concentrations are currently unavailable for AgNPs, and potential environmental exposures are poorly constrained, the increasing use of AgNPs has raised concerns over their likely release into ecosystems. Given the critical role of microbial communities in organic matter and nutrient cycling in ecosystems, environmental exposure of AgNPs has the potential to alter ecosystem productivity.13 A large number of studies have been conducted on the clinical applications of AgNPs,14–18 while their environmental impacts are largely unclear.12,19–25 In addition, the antimicrobial properties of complexes of AgNPs with

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conventional antibiotics that may form during their clinical use are completely unknown. Whether AgNPs pose a threat to microbes essential for recycling in natural or engineered systems is an outstanding question of great relevance to ecosystem health and sustainable nanotechnology. In the present study, we report the antimicrobial action of AgNPs and their complexes with the widely used antibiotics tetracycline and kanamycin on B. subtilis and Pseudomonas. These microbes play a critical role in elemental recycling, degradation of pollutants and plant growth.26 Strains of B. subtilis can convert explosives into the harmless compounds nitrogen, carbon dioxide, and water. They also play a vital role in radionuclide waste disposal due to the proton binding properties of their surfaces. Pseudomonas works as a biocontrol and bioremediation agent in nature. Certain members of the Pseudomonas genus prevent the growth or establishment of crop pathogens and metabolize chemical pollutants in the environment. In the present article, we report the synthesis of monodisperse AgNPs, tetracycline-adsorbed AgNPs and kanamycinadsorbed AgNPs and their antimicrobial properties against B. subtilis and Pseudomonas. The antimicrobial activities of AgNPs, tetracycline-adsorbed AgNPs and kanamycin-adsorbed AgNPs were evaluated by micro-dilution and disk diffusion methods. Minimum inhibitory concentrations (MIC) of AgNPs against both the organisms are 25 mg mL1, which is quite low. The additive effect of antibiotics is evidenced in the disk diffusion test. The zone of inhibition (ZIH) increases by 33 and 100%, respectively, when tetracycline and kanamycin are adsorbed on AgNPs. These results indicate that the non-target effects of AgNPs are enhanced when antibiotics are adsorbed onto them. Medical waste containing AgNPs could be more devastating to ecological systems compared to AgNPs leached from other commercial nanotechnology based products.

Experimental section Materials Silver nitrate (99.8%) and diphenyl ether were purchased from S D Fine-Chem Limited. Oleylamine (70%) and pluronic F-127 were obtained from Sigma Aldrich. Absolute ethanol, n-hexane (95%) and HPLC grade water were obtained from Merck. Mueller Hinton Agar (MM019) and nutrient broth (NM019) were purchased from Sisco Research Laboratories. All the chemicals were used as received without any further purication. The cultures Bacillus subtilis and Pseudomonas were procured from IMTEC, Chandigarh, India. The antibiotics tetracycline hydrochloride and kanamycin monosulphate were purchased from Himedia. Synthesis of silver nanoparticles AgNPs can be prepared by a variety of methods such as thermal decomposition,27 microemulsion,28 sol–gel,29 green synthesis using microbes,30 thermal, radiation and chemical reduction methods,27,31–34 etc. Reduction in the presence of a mild reducing agent is the most promising approach for the

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synthesis of AgNPs .35 In the present study, the preparation of uniform, monodisperse AgNPs was carried out by a simple onepot method. In brief, a 100 mL 3-necked round bottom ask (RBF), equipped with a magnetic stirrer, condenser and a thermometer, was lled with 20 mL diphenyl ether and 15 mM oleylamine. The mixture was heated to 200  C at a rate of 3  C min1. In the above mixture, 3 mM AgNO3 was added under continuous magnetic stirring. Upon addition of AgNO3, the color of the mixture immediately turned blue. Strong surface plasmon resonance (SPR) was observed, indicating the nucleation of AgNPs. The mixture was reuxed at this temperature for 30 min and then rapidly cooled to 150  C. It was then agitated and ripened at 150  C for another 4 h and then cooled to room temperature. The product was puried by precipitation and redispersion in ethanol and n-hexane, respectively.36 The coating of oleylamine on the surface of AgNPs renders them hydrophobic. To investigate the antimicrobial activities of AgNPs, they must be water dispersible. Hence, the as-synthesized hydrophobic AgNPs were phase transferred from n-hexane to water by facile phase transfer protocols.36 In brief, an equal volume of an aqueous solution of pluronic F-127 was mixed with the colloidal dispersion of AgNPs in n-hexane. The concentration of the pluronic F-127 was kept such that the Ag : pluronic F-127 weight ratio was 1 : 0.5. At the beginning of the phase transfer, the organic phase of AgNPs is well separated from the aqueous phase of pluronic F-127, and upon magnetic stirring both immiscible phases mix with each other. The mixture was covered with a perforated aluminium foil to control the evaporation of n-hexane. It was magnetically stirred until the organic phase evaporated completely. To conrm the phase transfer of AgNPs, an equal volume of fresh n-hexane was poured into the aqueous solution of AgNPs. Upon successful phase transfer, both aqueous and organic phases would remain immiscible. Antibiotic-adsorbed AgNPs were prepared by vortex mixing of 10 mL aqueous colloidal solution of AgNPs with the desired quantity of tetracycline and kanamycin for 12 h. The nal concentration of AgNPs in antibiotic-adsorbed AgNPs was adjusted to 250 ppm. The mixture was centrifuged at 12 000 rpm, the supernatant was removed, and the nal volume of the particles was readjusted to 10 mL by the addition of ultrapure water. This stock solution of antibiotic-adsorbed AgNPs were further used for the antibacterial activity tests.

Characterization of AgNPs Structural characterization of AgNPs was carried out by recording their powder X-ray diffraction pattern. The XRD pattern was recorded on a PANalytical X'Pert Pro diffractometer operated at 40 kV and 45 mA at 25  C with monochromatic CuKa radiation of wavelength 0.15406 nm. HRTEM micrographs and selected area electron diffraction (SAED) patterns were recorded on a JEOL (Model JEM 2100F) transmission electron microscope operated at an accelerating voltage of 200 kV. The sample for TEM microscopy was prepared by placing a drop of the colloidal dispersion of AgNPs in n-hexane onto an amorphous carboncoated copper grid, and n-hexane was allowed to evaporate

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slowly at room temperature. The particle size distribution histogram was prepared by measuring the diameter of 100 particles from the HRTEM micrograph. The hydrodynamic sizes and size distribution of AgNPs were obtained by photon correlation spectroscopy. The measurements were carried out on a Brookhaven 90 plus Particle Size Analyzer at 25  C. To conrm the formation of AgNPs, UV-visible spectra were recorded on a Hitachi U-3900H double beam UV-visible spectrophotometer. The spectra were recorded from 300–600 nm at 25  C. The concentration of Ag was determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). The measurements were performed on a Spectro ARCOS model. The sample for ICP measurement was prepared by digestion of the aqueous colloidal AgNPs in nitric acid. Antibiotic content and antibiotic loading The antibiotic content and antibiotic loading efficiency was determined by UV-visible spectroscopy. The optical density of antibiotics that remained unincorporated (supernatant) into the AgNPs was measured at 357 nm for tetracycline and at 256 nm for kanamycin, and the concentration was determined by the Beer–Lambert law, A ¼ 3cl, where A is the optical density at sample concentration c, l is the path length of the sample cell (10 mm) and 3 is the molar absorptivity of the drug (3 for tetracycline and kanamycin is 14.58  106 M1 cm1 and 143.40 M1 cm1, respectively). Antibiotic content and antibiotic loading efficiency were determined by using the following expressions,37 Antibiotic content ¼ weight of antibiotic adsorbed on nanoparticles  100 total weight of nanoparticles

Antibiotic loading efficiency ¼ [(W1  W2)/W1]  100 where W1 is the total weight of the antibiotic and W2 is the weight of the free antibiotic, which was not adsorbed onto the nanoparticles (NPs).

Environmental Science: Processes & Impacts

suspension (approximately 1  108 colony forming units (CFU) per mL). The inoculated sets were incubated at 37  C for 24 h. The experiments were carried out in triplicate. Evaluation of the MIC was performed by visual inspection of growth/no growth in mixtures containing different concentrations of AgNPs. The lowest concentration of Ag that inhibited bacterial growth was taken as the MIC for that particular bacterium. Control experiments were also run in parallel to investigate the antibacterial activities of nutrient broth medium. In the disk diffusion test, Mueller–Hinton Agar medium was prepared as per the standard protocols by dissolving 39 g Mueller–Hinton Agar in 1 L of distilled water. 20 mL of Mueller Hinton Agar medium was added to the disposable Petri dishes and allowed to solidify. The bacterial strain (108 CFU mL1) was inoculated onto the entire surface of a Mueller–Hinton agar plate with the help of a sterile cotton-tipped swab to form an even lawn. A sample disk containing 50 mL of the antibacterial agent (AgNPs/tetracycline/kanamycin/tetracycline-adsorbed AgNPs/kanamycin-adsorbed AgNPs) was put on every disk. Each set was incubated at 37  C for 24 h. The zone of inhibition was measured aer the incubation period. Each experiment was carried out in triplicate and the results were statistically averaged.

Results The X-ray diffraction pattern of as-synthesized AgNPs is shown in Fig. 1. The diffraction pattern shows four well resolved diffraction peaks corresponding to the standard FCC structure of Ag (PDF card no. 040783). The crystallite size of the AgNPs was determined from the highest intense reection (111) by using the Scherrer equation, and it was found to be 3.3 nm. Fig. 2 shows representative HRTEM micrographs of the assynthesized AgNPs. Nanocrystals of Ag self-assembled into a regular hexagonal closed packed lattice. Each particle has spherical or near spherical morphology. No agglomeration was observed. In order to measure the physical size, a size

Antibacterial activity of AgNPs The antimicrobial activities of AgNPs have been evaluated by measuring the minimum inhibitory concentration (MIC) of the nanostructures against environmentally friendly (B. subtilis and Pseudomonas) microorganisms. The MIC is the concentration required to completely inhibit the bacterial growth. The MIC values were determined by a micro-dilution method. Antimicrobial activities were also measured by a disk diffusion test, in which the zone of inhibition of microorganisms was measured on an agar plate. The zone of inhibition is the area on the agar plate where the bacterial growth is prevented by antimicrobial agents. In the micro-dilution method, six sets of 10 mL nutrient broth medium containing AgNPs with effective Ag concentrations of 0 to 200 mg mL1 were prepared. Each set was inoculated aseptically with 10 mL of the respective bacterial This journal is © The Royal Society of Chemistry 2014

XRD pattern of as-synthesized AgNPs. Four diffraction peaks are seen, corresponding to the standard FCC structure of Ag.

Fig. 1

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UV-visible spectra of AgNPs (-- --) before and (—) after phase transfer. A single SPR peak in both the curves indicates the spherical geometry of NPs. The SPR band is red shifted from 404 nm to 413 nm after phase transfer.

Fig. 4

Fig. 2 HRTEM image of the as-synthesized AgNPs. Inset (top) shows the specific area electron diffraction pattern (SAED) and inset (bottom) shows the size distribution histogram, fitted with a lognormal size distribution function. Well resolved diffraction rings observed in the SAED pattern correspond well with the X-ray diffraction peak index in Fig. 1. The polycrystalline nature of the NPs is confirmed from the SAED ring pattern.

distribution histogram was constructed by measuring the diameter of at least 100 particles. The histogram is tted with the lognormal size distribution function38 (inset of Fig. 2). The average physical size and polydispersity index obtained from the t are 8.07  0.02 nm and 0.17  0.003, respectively. The low value of the polydispersity index conrms our belief that the synthesized nanostructures are nearly monodisperse in size. A representative SAED image of the AgNPs is also shown in the inset of Fig. 2. Four well resolved diffraction rings corresponding to the (111), (200), (220) and (311) diffraction planes of the FCC crystal lattice were observed. The hydrodynamic size distribution histograms of AgNPs recorded before and aer the

phase transfer are shown in Fig. 3. Histograms were tted with a lognormal size distribution function38 and the mean hydrodynamic sizes were found to be 10.2 nm and 59 nm, respectively. The mean hydrodynamic size of AgNPs before phase transfer is greater than that of the corresponding crystallite size and physical size obtained from X-ray diffraction and high resolution transmission electron microscopy, respectively. This is quite obvious as the hydrodynamic size accounts for both the core and the polymeric coating, while TEM and X-ray techniques do not account for any polymer present on the surface of the NPs. The UV-visible spectra of the AgNPs recorded before and aer the phase transfer are shown in Fig. 4. A single plasmon peak was observed in both spectra, which indicates that the particle morphology is not affected by the phase transfer. A slight red shi is observed in the spectral band aer the phase transfer. This might be due to the difference in surface adsorbed species on the AgNPs before and aer the phase transfer.31 Adsorption of antibiotics on AgNPs

Fig. 3 Size distribution histograms obtained from photon correlation spectroscopy of AgNPs (a) before and (b) after phase transfer. Each histogram is fitted with a lognormal distribution function.

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To understand the interaction of antibiotics with AgNPs, the UV-visible spectra of AgNPs, tetracycline, kanamycin, tetracycline-adsorbed AgNPs and kanamycin-adsorbed AgNPs were recorded. Absorption spectra corresponding to these samples are shown in Fig. 5. The characteristic SPR band of AgNPs is observed at 428.5 nm. The UV-visible spectrum of tetracycline shows three characteristic absorption bands centered at 357 nm, 275 nm and 250 nm, respectively. These correspond to the p / p* transitions of C]C. Kanamycin does not show any characteristic absorption bands in the UV-visible region of the electromagnetic spectrum. However, its complex with copper shows characteristic absorption at 256 nm, which is also shown in Fig. 5. In order to understand the interaction of tetracycline and kanamycin with AgNPs, UV-visible spectra of tetracyclineadsorbed AgNPs and kanamycin-adsorbed AgNPs were also recorded. The peaks of tetracycline are now observed at 374 nm, 272 nm and 252 nm, respectively. The SPR band originating from the AgNPs in the tetracycline–AgNPs complex shows a red

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Antibacterial activity

UV-visible spectra of (a) AgNPs (b) tetracycline (c) kanamycin (d) tetracycline-adsorbed AgNPs and (e) kanamycin-adsorbed AgNPs. The inset shows a magnified view of the UV-visible spectra of (a) AgNPs (d) tetracycline-adsorbed AgNPs and (e) kanamycin-adsorbed AgNPs.

Fig. 5

shi and is positioned at 435 nm. In the case of kanamycinadsorbed AgNPs, the characteristic absorption of kanamycin at 256 nm shis to 252 nm while the SPR band of AgNPs is shied from 428 nm to 430 nm. A slight blue shi in the spectral vibrations of tetracycline, kanamycin and AgNPs indicates that the antibiotics are adsorbed onto the surface of the AgNPs. Antibiotic content and antibiotic loading Antibiotic content and antibiotic loading efficiency were also determined by UV-visible spectroscopy. The loading efficiency of tetracycline is found to be 15.3%, while the drug content in the antibiotic-adsorbed AgNPs system is 40.56%. For kanamycin, the loading efficiency is measured as 6.44% and the drug content in the antibiotic-adsorbed AgNPs system is 90.40%.

Antimicrobial activity studies of AgNPs were performed against two environmentally friendly bacteria (B. subtilis and Pseudomonas) by micro-dilution and disk diffusion tests. From the micro-dilution method, the MIC values of as-synthesized AgNPs were determined for these bacterial strains. To further understand the effectiveness of AgNPs against these environmentally friendly microorganisms, the disk diffusion test was also carried out in the presence of commercially available antibiotics; tetracycline and kanamycin. In this test, the zone of inhibition was measured aer incubating the strains in the presence of AgNPs for 24 h. The results of the MIC tests are presented in Fig. 6. The MIC value of both the microorganisms is found to be 25 mg mL1. It is clearly evidenced that the MIC values of AgNPs for environmentally friendly microorganisms are quite low as compared to those for pathogenic microorganisms.39,40 This observation indicates that the environmentally friendly microorganisms under study are more susceptible to AgNPs as compared to pathogenic strains. The results of antimicrobial tests of AgNPs by the disk diffusion method are shown in Table 1. The inhibition zone for these environmentally friendly microorganisms was found to be in the range of 6–7 mm. To understand the additive effects of AgNPs, the disk diffusion experiments were also carried out for tetracycline-adsorbed AgNPs and kanamycin-adsorbed AgNPs. Control experiments containing tetracycline and kanamycin only were also run parallel to these experiments. The images of the disk diffusion tests are presented in Fig. 7. An additive effect of AgNPs is clearly evidenced by the increase in the zone of inhibition with respect to the inhibition zone found for a particular antibiotic. A sharp increase in the inhibition zone was observed when antibiotics were used in conjunction with AgNPs. The inhibition zone increases by 33.33% and 100%, respectively, when tetracycline and kanamycin were tested with

Fig. 6 Photographic view of MIC tests of AgNPs on B. subtilis and Pseudomonas. Order of the tubes from left to right (1). Negative control (i.e., medium alone); (2). Control (i.e., medium + antibacterial agent (AgNPs)); (3). Zero concentration (i.e., medium + respective bacterial strain); (4). 30% below MIC (i.e., medium + respective bacterial strain + AgNPs at a concentration 30% below the MIC); (5). 10% below MIC (i.e., medium + respective bacterial strain + AgNPs at a concentration 10% below the MIC); (6). At MIC (i.e., medium + respective bacterial strain + AgNPs at a concentration equal to the MIC).

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Additive effect of AgNPs on the antibacterial activities of commercial antibiotics

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Zone of inhibition (ZIH) (in mm) Strains

Nature

Control

Tetracycline

Kanamycin

AgNPs

Tetracycline-adsorbed AgNPs

Kanamycin-adsorbed AgNPs

B. subtilis Pseudomonas

Gram positive Gram negative

0 0

40 30

12 10

6 7

50 40

20 30

Fig. 7 Zone of inhibition against environmentally friendly bacterial strains (1) B. subtilis (2) Pseudomonas, where C ¼ control, A ¼ tetracycline, B ¼ kanamycin, P ¼ AgNPs, AP ¼ tetracycline-adsorbed AgNPs, BP ¼ kanamycin-adsorbed AgNPs.

AgNPs against Pseudomonas. Against B. subtilis, the inhibition zone increases by 25% and 66.7% for tetracycline- and kanamycin-adsorbed AgNPs, respectively.

Discussion The synthesis of AgNPs was carried out via a two-step procedure. In the rst step, AgNPs were produced by reducing AgNO3 with oleylamine, and in the second step, the as-synthesized hydrophobic AgNPs were phase transferred into an aqueous medium by using a triblock co-polymer, pluronic F-127. For homogeneous nucleation of Ag from AgNO3 by oleylamine, it is necessary to set the nucleation temperature at $150  C.36 Therefore, in order to achieve fast and homogenous nucleation, AgNO3 was added into the preheated mixture of oleylamine–diphenyl ether. The reaction temperature was lowered to 150  C, as ripening at low temperature will give better control over the growth of individual crystallites.39 To study the antimicrobial activities, water dispersible NPs are required. Phase transfer protocols were developed to transfer hydrophobic NPs to hydrophilic by using pluronic F127 as a transfer ligand. The phase transferred AgNPs exhibit good stability in water. These water dispersible AgNPs have similar physical properties to those dispersed in n-hexane (Fig. 4). The only difference is in the hydrodynamic size (Fig. 3). The hydrodynamic sizes of particles before and aer phase transfer were 10.2 nm and 59 nm, respectively. The micellar diameter of pluronic F-127 is 23 nm.41 This means that a multilayer of pluronic F-127 is coated on the AgNPs. MIC and disk diffusion tests clearly indicate strong antimicrobial activities of tailored AgNPs against the non-pathogenic strains of B. subtilis and Pseudomonas, which are playing critical

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roles in maintaining the balance in nature. ZIH values demonstrated a lower antibacterial effect of AgNPs towards Gram-positive bacteria (B. subtilis) as compared with that towards a Gram-negative strain of Pseudomonas. Antibioticadsorbed AgNPs also show similar results. This may be due to the fact that AgNPs may penetrate more easily through the outer membrane of Gram-negative bacteria than the thick peptidoglycan layer of Gram-positive bacteria. The mechanism of bactericidal action of AgNPs is not well understood. There are, however, various theories on the action of AgNPs on bacterial strains to cause the antibacterial effect. AgNPs may attach to the surface of the cell membrane and disturb the permeability and respiration of bacteria. Ag ions are released from AgNPs and react with thiol groups of proteins and interfere with DNA replication.42 DNA loses its replicative ability and expression of ribosomal subunits when the microorganisms are exposed to Ag ions. The bacterial membrane also becomes damaged by the free radicals generated by AgNPs. The direct physical contact between AgNPs and bacterial cells causes structural damage to the cell wall, which nally leads to cell death. The toxicity of AgNPs could be based on the available exposed surface for reaction with the cell, which increases with the decrease in size of the particles. Some researchers suggest that AgNPs may also pass through the cell wall of bacteria to oxidize the surface proteins on the plasma membrane and consequently disturb the cell homeostasis. Oxidative stress is an important mechanism of DNA damage.43 A bacterial cell in contact with AgNPs takes in Ag ions. These ions can inhibit respiratory enzymes leading to reactive oxygen species formation, which results in damage to the cell.44 Ag ions also affect the function of membrane-bound enzymes, such as those in the respiratory chain. Sulfur containing proteins in the membrane or inside the cells and phosphorus containing elements such as DNA are likely to be the preferential sites for the binding of AgNPs. Ag has a greater tendency to react with sulfur- or phosphoruscontaining so bases.45 Disruption of membrane morphology may cause a signicant increase in permeability, leading to uncontrolled transport through the plasma membrane and, nally, cell death. One or more of these possible mechanisms might be responsible for the strong antimicrobial action of our tailored AgNPs.

Conclusions Nearly monodisperse self-assembled AgNPs are prepared via a chemical reduction method and the commercially available antibiotics tetracycline and kanamycin are adsorbed onto the

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AgNPs. The antibacterial action of antibiotic-adsorbed AgNPs on the biorecycling microbes B. subtilis and Pseudomonas is increased by 33–100% when compared with the antibacterial action of AgNPs alone. This study shows that environmental exposure of antibiotic-adsorbed AgNPs leached from medical waste could be much more potent towards biorecycling microbes in comparison to AgNPs leached from other domestic nanotechnology based products.

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Acknowledgements CK is thankful to the Council of Scientic and Industrial Research, New Delhi for a senior research fellowship (Scheme no. 03(1226)/12/ERM-II). BC acknowledges the nancial support from the Department of Science & Technology, New Delhi under its research scheme (SR/FTP/PS-109/2010) and University Grants Commission (UGC), New Delhi under scheme (F. no. 42850/2013 (SR)). NA acknowledges the nancial support from the UGC, New Delhi under the DS Kothari postdoctoral fellowship scheme (F. 4-2/2006(BSR)/B-467/2011(BSR)). The authors are also thankful to Mr Rajan Singh for his help in HRTEM measurements.

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2198 | Environ. Sci.: Processes Impacts, 2014, 16, 2191–2198

This journal is © The Royal Society of Chemistry 2014

Antibacterial activities of silver nanoparticles and antibiotic-adsorbed silver nanoparticles against biorecycling microbes.

Silver nanoparticles have a huge share in nanotechnology based products used in clinical and hygiene products. Silver nanoparticles leaching from thes...
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