Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field

Transcription

1 Umadevi et al: Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field 1491 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 4 Issue 3 October December 2011 Research Paper MS ID: IJPSN UMADEVI Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field M. Umadevi 1 *, T. Rani 1, T. Balakrishnan 2 and R. Ramanibai 2 1 Department of Physics, Mother Teresa Women s University, Kodaikanal , Tamil Nadu, India. 2 Department of Zoology, University of Madras, Guindy Campus, Chennai , Tamil Nadu, India. Received July 07, 2011; accepted November 1, 2011 ABSTRACT Nanotechnology has great promise for improving the therapeutic potential of medicinal molecules and related agents. In this study, silver nanoparticles of different sizes were synthesized in an ultrasonic field using the chemical reduction method with sodium borohydride as a reducing agent. The size effect of silver nanoparticles on antimicrobial activity were tested against the microorganisms Staphylococcus aureus (MTCC No. 96), Bacillus subtilis (MTCC No. 441), Streptococcus mutans (MTCC No. 497), Escherichia coli (MTCC No. 739) and Pseudomonas aeruginosa (MTCC No. 1934). The results shows that B. subtilis, and E. coli were more sensitive to silver nanoparticles and its size, indicating the superior antimicrobial efficacy of silver nanoparticles. KEYWORDS: Optical absorption; silver NP; particle size analyzer; antimicrobial activity; size effect. Introduction Silver and silver ions have long been known to have strong inhibitory and antibacterial effects (Kim et al., 1998). The resurgence of the use of silver-based antiseptic materials may be linked to a broad spectrum of activity and a lower propensity to induce microbial resistance than antibiotics (Jones et al., 2004). Many researchers have tried to measure the activity of metal ions against microorganisms. Even though copper and zinc show antimicrobial properties, these nanoparticles have limited usefulness as an antimicrobial agent for several reasons, including the interfering effects of salts and the antimicrobial mechanism. These limitations can be overcome with silver nanoparticles, which exhibit efficient antimicrobial property compared to other salts due to their large surface area providing better contact with microorganisms. The nanosize space allowed expansion of the contact surface of silver with the microorganisms and this nanoscale has applicability for medical devices as surface-coating agents (Kim et al., 2004). The nano-silver can inhibit the growth of a wide variety of microorganisms. Recently, health care providers and researchers took a renewed interest in silver because the pathogens, when exposed, showed increased resistance capability to antibiotics. In addition, the nanoscale technique development for producing silver nanoparticles may assist medical use, especially in applications where fighting germs is a major concern. Silver nanoparticles which have a high specific surface area and a high fraction of surface atoms have attracted the attention of the industry because of their unique characteristics of high efficiency and antimicrobial activity even at low concentration volumes (Kowshik et al., 2003; Duran et al., 2005). Silver nanoparticles also possess low toxicity to human cells, high thermal stability and low volatility (Duran et al., 2007). Silver nanoparticles can be therefore be exploited in medicine for burn treatment, dental materials, coating stainless steel materials, textile fabrics, water treatment and sunscreen lotions. In the present study, silver nanoparticles of different sizes were prepared in an ultrasonic field. Antimicrobial activity of silver nano particles and its size effect have been tested against five different microorganisms Staphylococcus aureus (MTCC No. 96), Bacillus subtilis (MTCC No. 441), Streptococcus mutans (MTCC No. 497), Escherichia coli (MTCC No. 739) and Pseudomonas aeruginosa (MTCC No. 1934). Materials Materials and Methods Silver nitrate and sodium borohydride were obtained from Aldrich Chemicals, India. They were used as such without any further purification. 1491

2 1492 Int J Pharm Sci Nanotech Vol 4; Issue 3 October December 2011 Preparation of Silver Nano Particles The silver nanoparticles used in this study were synthesized by the chemical reduction method. In brief, 4 ml of silver nitrate solution was added rapidly to 25 ml of sodium borohydride solution in the ultrasonic field. It was repeated for different volume of silver nitrate (8, 12 and 16 ml) with constant volume of sodium borohydride solution (25 ml). Both solutions were chilled to ice temperature. The prepared silver nanoparticles show surface plasmon resonance around 395 nm (Table 1; Figure 1) :25 8:25 12:25 16:25 spread with 100 μl of actively grown broth cultures of the respective test bacteria and allowed to dry for 10 minutes. Sterile readymade discs loaded with each silver nanoparticle individually (15 μl/disc, 20 μl/disc and 25 μl/disc) were imposed on the inoculated plates. The plates were incubated for 48 hours at 37 o C. The development of the inhibition zone around the extract loaded discs was recorded. Antibiotic-impregnated discs released antibiotics into the surrounding medium when placed on the surface of solid agar. Untreated agar plates were inoculated with test microorganisms and antibiotic discs were placed on the agar surface. Disc diffusion is a qualitative method based on an approximation of the effect of antibiotic material on bacterial growth on solid medium. A zone of growth inhibition around the antibiotic will occur if the organism is susceptible to the antibiotic. As the distance from the disc increases, there is a logarithmic decrease in antibiotic concentration. As the zone size increases, the minimum inhibitory concentration (MIC) of the antibiotic decreases. Absorbance Apparatus Optical absorption spectra were recorded using a (Shimadzu UV 1700 Pharmaspec) UV visible spectrophotometer. Particle size distributions were made using a Nanotrac Ultra NPA253 particle size analyzer. The Nanotrac measurement technique is based on dynamic light scattering. 0.2 Results and Discussion wavelength / nm Fig. 1. Optical absorption spectra of silver nanoparticles (4:25 4 ml of AgNO3 in 25 ml of NaBH4). TABLE 1 Particle size of prepared silver nanoparticles. S. No. Volume of aqueous (ml) AgNO3:NaBH4 Surface Plasmon Resonance peak (nm) Particle size (diameter) (nm) 1 4: : : : Assay for Antimicrobial Activity of Silver Nanoparticles Against Microorganisms The antimicrobial activity of silver nanoparticles was evaluated against Staphylococcus aureus (MTCC No. 96), Bacillus subtilis (MTCC No. 441), Streptococcus mutans (MTCC No. 497), Escherichia coli (MTCC No. 739) and Pseudomonas aeruginosa (MTCC No. 1934) by the agar disc diffusion method. Mueller Hinton agar plates were Silver Nanoparticle Size Determination Gustav Mie was the first to provide an explanation on the dependence of color on the metal particle size (Bohren et al., 1983). When the size of the particle is comparable to their skin depth, all the electrons in the particle resonates, resulting in strong absorption of the particular wavelength. The color of the colloid depends not just on the particle size, but also on the shape, the refractive index of the surrounding media and the separation between the particles. A change in any of these parameters will result in the quantifiable shift in the surface plasmon resonance absorption peak (Valmalette et al., 1996). The optical properties of dispersions of spherical particles with a radius, r can be predicted by the Mie theory (Bohren et al., 1983). Using this Mie theory, the radius of the silver Nanoparticles had been calculated (Table 1). The absorption spectra of the silver nanoparticles is represented in Figure 1. The absorption spectrum of these samples depicted a well-defined surface plasmon band in the range of nm, which is a characteristic of nanosized silver. It further confirms the nanocrystalline character of the articles and the low degree of their polydispersity (Schneider et al., 1994).

3 Umadevi et al: Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field 1493 This absorption band results from interactions of free electrons confined to small metallic spherical objects with incident electromagnetic radiation. Electronic modes in silver nanoparticles were particularly sensitive to their shape and size, leading to pronounced effects in the visible part of the spectrum. The observed plasmon band indicated that the silver nanoparticles were spherical in shape (Mock et al., 2002). Figure 2 shows the particle size distribution. It is a histogram of silver nanoparticles prepared with a) 16 ml of AgNO3 in 25 ml of NaBH4 and b) 4 ml of AgNO3 in 25 ml of NaBH4. Figure 2A shows a maximum distribution at 1.9 nm and Figure 2B shows a maximum distribution at 5.37 nm. The histogram shows that as the volume of silver nitrate increases, silver nanoparticle size decreases. increase in particle size. The smaller the size of the nanoparticles, the larger the surface to volume ratio and a larger percentage of the bacterial cells will be adsorbed on the silver nanoparticles (Morones et al., 2005; Pal et al., 2007). The nanoparticles smaller than 10 nm interact with bacteria and produce electronic effects which enhance the reactivity of nanoparticles. Thus it is corroborated that the bactericidal effect of silver nanoparticles is size dependent (Morones et al., 2005). Silver nanoparticles may attached to the surface of the cell membrane and disturb its power functions such as permeability and respiration. It is reasonable to state that the binding of the particles to the bacteria depends on the surface area available for interaction. Small particles having a larger area available for interaction will give more bactericidal effect than the larger particles. In the present study, antimicrobial tests were performed against S. aureus, B. subtilis, S. mutans, E. coli and P. aeruginosa on Mueller Hinton agar plates treated with different volume of silver nanoparticles. The concentration depended on the antimicrobial efficacy of the silver nanoparticles of two different sizes on the above mentioned organisms were shown in Table 2A and 2B. TABLE 2A. Antimicrobial efficacy of silver nanoparticle of diameter 32 nm. Fig. 2A. Particle size histogram of silver nanoparticles prepared with 16 ml of AgNO3 in 25 ml of NaBH4. Organism Zone diameter (mm) 15 μl/disc 20 μl/disc 25 μl/disc Staphylococcus aureus -* -* -* Bacillus subtilis Streptococcus mutans Escherichia coli -* 7 9 Pseudomonas aeruginosa -* -* 7 TABLE 2B. Antimicrobial efficacy of silver nanoparticle of diameter 20 nm. Fig. 2B. Particle size histogram of silver nanoparticles prepared with 4 ml of AgNO3 in 25 ml of NaBH4. Antimicrobial Activity of Silver Nanoparticles Against Microorganisms Surface plasmon resonance plays a major role in the determination of optical absorption spectra of metal Nanoparticles, which shifts to longer wavelengths with Organism Zone diameter (mm) 15 μl/disc 20 μl/disc 25 μl/disc Staphylococcus aureus -* 7 7 Bacillus subtilis Streptococcus mutans Escherichia coli Pseudomonas aeruginosa -* -* 7 * No antimicrobial activity was found with the concentrations used in this work. The observed result showed that silver nanoparticles were most effective against E. coli. The minimal inhibitory concentration (MIC) values were calculated using an equation derived by Dimitriu et al., The MIC of silver nanoparticles against E. coli may be estimated between 6.73 mg/l, and 5.60 mg/l and the growth inhibition effect was observed in a volume dependent manner. In addition, as the size of the nanoparticles decreases, the antimicrobial activity increases.

4 1494 Int J Pharm Sci Nanotech Vol 4; Issue 3 October December 2011 In the case of P. areuginosa, the antimicrobial efficacy of silver nanoparticles is independent on particle size. In both the sizes of silver nanoparticles it exhibited inhibition only at 25 μl/disc. In S. aureus, silver nanoparticles showed a mild growth inhibitory effect even in high concentration. The silver nanoparticles having 32 nm diameter did not show any antimicrobial activity in all the three different volumes of silver nanoparticles. Silver nanoparticles with 20nm diameter indicated the same antimicrobial efficacy at 20 μl/disc and 25 μl/disc. MIC of silver nanoparticles against S. aureus was estimated to be 6.73 mg/l. For B. subtitis, the silver nanoparticles with 20 nm diameter showed a stronger inhibition effect when compared to 32 nm silver nanoparticles. In both cases, as the volume of nanoparticles increases the antimicrobial effect also increases. In the case of S. mutans, 15 μl/disc and 20 μl/disc show same antimicrobial efficacy and it does not depend on the size of the particles. For 25 μl/disc, 20 nm silver nanoparticles show higher effect when compared to 32 nm silver nanoparticles. The exact mechanism of action of silver on the microbes is still not known but the possible mechanism of action of metallic silver, silver ion and silver nanoparticles had been suggested according to the morphological and structural changes found in the bacterial cells. The observed inhibition may be due to the following reasons. The positive charge on the silver ion is crucial for its antimicrobial activity through electrostatic attraction between negative charged cell membranes of microorganisms and positive charged nanoparticles (Hamouda et al., 2001; Dibrov et al., 2002). The antimicrobial effects of silver nanoparticles may be associated with characteristics of certain bacterial species. Gram-positive and gram negative bacteria had differences in their membrane structure, the most distinctive of which is the thickness of the peptidoglycan layer. The peptidoglycan layer is a specific membrane feature of bacterial species and not mammalian cells. Peptidoglycan is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of bacteria, forming a cell wall. It serves a structural role in the bacterial cell wall, giving structural strength as well as counteracting the osmotic pressure of the cytoplasm. The peptidoglycan layer is thicker in Gram-positive bacteria than Gram-negative bacteria. Gram-positive bacterium has a thick cell wall with multiple peptidoglycan layers. It does not contain an outer membrane, periplasmic space or lipopolysaccharide. Instead, it possesses long chain teichoic acids interwined among the peptidoglycan. The teichoic acid is covalently attached to the peptidoglycan. Gram-negative bacterium, on the other hand, has a single thin peptidoglycan layer and no teichoic acids. It possesses an outer membrane containing lipopolysaccharide and a periplasmic space between the outer and inner membrane. The peptidoglycan is covalently attached to lipoprotein molecules which project into the outer membrane. The inhibition zone diameters of B. subtilis were larger than those of E. coli (32 nm diameter). This implies that silver had better antimicrobial effects against B. subtilis Gram-positive bacteria than E. coli Gram-negative bacteria. The lower sensitivity of gramnegative bacterial strains could probably be explained by the biochemical and physiological characteristics of those bacteria. It is well known that the outer membrane of Gram-negative bacteria is predominantly constructed from tightly packed lipopolysaccharide molecules which provide an effective resistive barrier (Sondi et al., 2004; Papo et al., 2005; Yoon et al., 2008). P. aeruginosa is a highly virulent opportunistic pathogen. One of the most worrisome characteristics of P. aeruginosa is its low antibiotic susceptibility. This low susceptibility is attributable to a concerted action of multi drug efflux pumps with chromosomally encoded antibiotic resistance genes and the low permeability of the bacterial cellular envelopes. In addition to this intrinsic resistance, P. aeruginosa easily develops acquired resistance either by mutation in chromosomally encoded genes or by the horizontal gene transfers of antibiotic resistance determinants. The phenotypic resistance associated to biofilm formation or to the emergence of small colony variants may be important in the response of P. aeruginosa populations to antibiotics treatment. The observed lower efficacy of the silver Nanoparticles against P. aeruginosa may derive from the difference as a point of membrane structure. Therefore if the antibacterial effect of silver Nanoparticles is associated with the peptidoglycan layer, it will be easier and more specific to use silver nanoparticles as an antimicrobial agent. S. mutans is the principal microbiological agent in the etiology of dental caries. Various methods have been used in the attempt to diminish colonization of this microorganism: from topical application of substances on the dental structure and DNA plasmids resistant to S. mutans, to the application of vaccines and antibodies against this microorganism (Sierra et al., 2008). Silver has an important antimicrobial effect which is dependent on superficial contact, in that silver can inhibit enzymatic systems of the respiratory chain and alter DNA synthesis. Silver nanoparticles may be the most effective for controlling S. mutans and the observed efficacy is independent of size of the nanoparticles in 15 and 20 μl/disc. The lower concentration could avoid staining the tooth. The observed mild inhibitory effect of Nanoparticles against S. aureus may be due to their membrane structure especially the thickness of the peptidoglycan layer (Kim et al., 2007). S. aureus are surrounded by a cytoplasmic membrane with a bilayer structure and a thick, bag shaped cell wall. The cell wall consists of peptidoglycans and polyol phosphate polymers known as teichoic acids (Bhagavan, 1992). S. aureus contains two

5 Umadevi et al: Antimicrobial Activity of Silver Nanoparticles Prepared Under an Ultrasonic Field 1495 different sortases which anchor proteins to peptidoglycan. The anchored proteins are responsible for the manifestation of infections. Overall differences in pathogenicity of S. aureus are attributed to genome islands encoding a lot of toxins (Pertica et al., 2008). This virulent and opportunistic pathogen is resistant to most powerful antibiotics. The antimicrobial activity of silver nanoparticles on gram negative bacteria was dependent on the concentration of silver Nanoparticles and was closely associated with the formation of pits in the cell wall of bacteria. A bacterial membrane with this morphology exhibits a significant increase in permeability, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane and resulting in cell death (Sondi et al., 2004). It is well known that the outer membrane of E. coli cells is predominately constructed from tightly packed lipopolysaccharide molecules, which provide an effective permeability barrier. The metal depletion may cause the formation of irregularly shaped pits in the outer membrane and change membrane permeability, which is caused by progressive release of lipopolysaccharide molecules and membrane proteins (Amro et al., 2000). This also may cause the degradation of the membrane structure of E. coli during treatment with silver nanoparticles (Sondi et al., 2004). Although their inference involved some sort of binding mechanism, still unclear is the mechanism of the interaction between silver nanoparticles and components of the outer membrane. The antimicrobial mechanism of silver nanoparticles is related to the formation of free radicals and subsequent free radical induced membrane damage. These free radicals may be derived from the surface of silver nanoparticles and are responsible for the antimicrobial activity (Kim et al., 2007). The MIC of all samples is lower when tested against E. coli than S. aureus. These results can be explained on the basis of the differences on the cellular wall of each strain. The cellular wall of gram-positive strains is wider than the cellular wall for Gram negative strains (Castanon et al., 2008). It is believed that DNA loses its replication ability and cellular protein becomes inactivated upon silver ion treatment (Feng et al., 2000). In addition, it was also shown that silver ion binds to functional groups of proteins, resulting in protein denaturation (Spadaro et al., 1974). Silver ions strongly interact with the available SH groups of the biomolecules to inactivate the bacteria. Furthermore, the antibacterial activity of silver ion under anaerobic conditions was found less potent than in oxygen-rich environments. Such interactions in the cell membrane would prevent DNA replications which would lead to bacterial death (Matsumura et al., 2003). In addition to the silver nanoparticles attached to the cell membrane, it also goes inside the bacteria (Morones et al., 2005). This suggested the possibility that the silver nanoparticles may also penetrate inside the bacteria and cause damage by interacting with phosphorus and sulphur containing compounds such as DNA. Conclusion Silver nanoparticles of different sizes have been synthesized by the chemical reduction method using sodium borohydride as reducing agent in an ultrasonic field. Size effect of silver nanoparticles on antimicrobial activity have been tested against the microorganisms S. aureus, B. subtilis, S. mutans, E. coli and P. aeruginosa. Silver Nanoparticles were most effective against B. subtilis and E. coli and seemed to be very sensitive to size of the nanoparticles. Acknowledgement The authors are thankful to Metrohm India Ltd, Chennai for analyzing particle size using Nanotrac facility. References Amro NA, Kotra LP, Mesthrige KW, Bulychev A, Mobashery S and Liu G (2000). High-Resolution Atomic Force Microscopy Studies of the Escherichia coli Outer Membrane: Structural Basis for Permeability. Langmuir 16: Bhagavan NV (1992). Medical Biochemistry 3 rd ed. Jones and Bartlett Publisher. Bohren CF and Huffman DF (1983). Absorption and scattering of light by small particles. Wiley, New York. Castanon GAM, Martinez NN, Gutierrez FM, Mendoza JRM and Ruiz F (2008). Synthesis and antibacterial activity of silver nanoparticles with different sizes. J Nanopart Res 10: Dibrov P, Dzioba J, Gosink KK and Hase CC (2002). Chemiosmotic Mechanism of Antimicrobial Activity of Ag + in Vibrio cholera. Antimicrob Agents Chemother 46: Dimitriu G, Poiata A, Tuchilus C and Buiuc D (2006). Correlation between linezolid zone diameter and minimum inhibitory concentration values determined by regression analysis. Rev Med Chir Soc Med Na Iasi 110: Duran N, Marcarto PD, De Souza GIM, Alves OL and Esposito E (2007). Antibacterial Effect of Silver Nanoparticles Produced by Fungal Process on Textile Fabrics and Their Effluent Treatment. J Biomed Nanotech 3: Duran N, Marcato PD, Alves OL and Souza G (2005). Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. J Nanobiotech 3:8 Feng QL, Wu J, Chen GQ, Cui FZ, Kim TM, and Kim JO (2000). A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 52: Hamouda T, Myc A, Donovan B, Shih A, Reuter JD, and Baker Jr JR (2001). A novel surfactant nanoemulsion with a unique nonirritant topical antimicrobial activity against bacteria, enveloped viruses and fungi. Microbiol Res 156:1-7. Jones SA, Bowler PG, Walker M, and Parsons D (2004). Controlling wound bioburden with a novel silver-containing Hydrofiber dressing. Wound Repair Regeneration 12: Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Kim SH, Park YK, Park YH, Hwang CY, Kim YK, Lee YS, Jeong DH, and Cho MH (2007). Antimicrobial effects of silver nanoparticles. Nanomedicine 3:

6 1496 Int J Pharm Sci Nanotech Vol 4; Issue 3 October December 2011 Kim TN, Feng QL, Kin JO, Wu J, Wang H and Chen GC (1998). Antimicrobial effects of metal ions (Ag+, Cu2+, Zn2+) in hydroxyapatite. J Mater Sci Mater Med 9: Kowshik M, Ashtaputre S and Kharrazi S (2003). Extracellular synthesis of silver nanoparticles by a silver-tolerant yeast strain MKY3. Nanotech 14: Matsumura Y, Kuniaki Y, Kunisaki SI, and Tsuchido T (2003). Mode of Bactericidal Action of Silver Zeolite and Its Comparison with That of Silver Nitrate. Appl Environ Microbiol 69: Mock JJ, Barbic M, Smith DR, Schultz DA and Schultz S (2002). Shape effects in plasmon resonance of individual colloidal silver nanoparticls. J Chem Phys 116: Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri J, Ramirez JT, and Yacaman MJ (2005). The bactericidal effect of silver nanoparticles. Nanotech 16: Pal S, Tak YK and Song JM (2007). Does the Antibacterial Activity of Silver Nanoparticles Depend on the Shape of the Nanoparticle? A Study of the Gram-Negative Bacterium Escherichia coli. Appl Environ Microbiol 73: Papo N and Shai Y (2005). A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides. J Biol Chem 280: Pertica A, Gavriliu S, Lungu M, Buruntea N and Panzaru C (2008). Colloidal silver solutions with antimicrobial properties. Mat Sci Eng 152: Schneider S, Halbig P, Grau H, and Nicket U (1994). Reproducible preparation of silver sols with uniform particle size for application in surface-enhanced raman spectroscopy. Photochem Photobiol 60: Sierra JFH, Ruiz F, Pena DCC, Gutierrez FM, Martinez AE, Guillen AJP, Perez HT and Castanon GM (2008). The antimicrobial sensitivity of Streptococcus mutans to nanoparticles of silver, zinc oxide, and gold. Nanomedicine 4: Sondi I and Sondi BS (2004). Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J Colloid Interface Sci 275: Spadaro JA, Berger TJ, Barranco SD, Chapin SE, and Becker RO (1974). Antibacterial Effects of Silver Electrodes with Weak Direct Current, Microb Agents Chemother 6: Valmalette JC, Lemaire L, Hornyak GL, Dutta J and Hofmann H (1996). Analysis Magazine 24. Yoon KY, Byeon JH, Park CW and Hwang J (2008). Antimicrobial Effect of Silver Particles on Bacterial Contamination of Activated Carbon Fibers. Environ Sci Technol 42: Address correspondence to: Dr. M. Umadevi, Associate Professor, 1Department of Physics, Mother Teresa Women s University, Kodaikanal , Tamil Nadu, India. Mob: ; ums10@yahoo.com