Elsevier

Acta Biomaterialia

Volume 4, Issue 3, May 2008, Pages 707-716
Acta Biomaterialia

Strain specificity in antimicrobial activity of silver and copper nanoparticles

https://doi.org/10.1016/j.actbio.2007.11.006Get rights and content

Abstract

The antimicrobial properties of silver and copper nanoparticles were investigated using Escherichia coli (four strains), Bacillus subtilis and Staphylococcus aureus (three strains). The average sizes of the silver and copper nanoparticles were 3 nm and 9 nm, respectively, as determined through transmission electron microscopy. Energy-dispersive X-ray spectra of silver and copper nanoparticles revealed that while silver was in its pure form, an oxide layer existed on the copper nanoparticles. The bactericidal effect of silver and copper nanoparticles were compared based on diameter of inhibition zone in disk diffusion tests and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of nanoparticles dispersed in batch cultures. Bacterial sensitivity to nanoparticles was found to vary depending on the microbial species. Disk diffusion studies with E. coli and S. aureus revealed greater effectiveness of the silver nanoparticles compared to the copper nanoparticles. B. subtilis depicted the highest sensitivity to nanoparticles compared to the other strains and was more adversely affected by the copper nanoparticles. Good correlation was observed between MIC and MBC (r2 = 0.98) measured in liquid cultures. For copper nanoparticles a good negative correlation was observed between the inhibition zone observed in disk diffusion test and MIC/MBC determined based on liquid cultures with the various strains (r2 = −0.75). Although strain-specific variation in MIC/MBC was negligible for S. aureus, some strain-specific variation was observed for E. coli.

Introduction

Microbial contamination of water poses a major threat to public health. With the emergence of microorganisms resistant to multiple antimicrobial agents [1] there is increased demand for improved disinfection methods. The antimicrobial properties of silver ions were known since ancient times and silver ions are widely used as bactericide in catheters, burn wounds and dental work [2]. Researchers have also recommended the use of silver and copper ions as superior disinfectants for wastewater generated from hospitals containing infectious microorganisms [3], [4]. However, residual copper and silver ions in the treated water may adversely affect human health [5]. The emergence of nanoscience and nanotechnology in the last decade presents opportunities for exploring the bactericidal effect of metal nanoparticles. The bactericidal effect of metal nanoparticles has been attributed to their small size and high surface to volume ratio, which allows them to interact closely with microbial membranes and is not merely due to the release of metal ions in solution [6]. Metal nanoparticles with bactericidal activity can be immobilized and coated on to surfaces, which may find application in various fields, i.e., medical instruments and devices, water treatment and food processing. Metal nanoparticles may be combined with polymers to form composites for better utilization of their antimicrobial activity. Metal nanoparticles are also finding application in various other fields, i.e., catalysis and sensors [7], [8], [9]. However, it is also recognized that nanoparticles may have many undesirable and unforeseen effects on the environment and in the ecosystem [10], [11].

The antimicrobial properties of silver nanoparticles are well-established [12], [13], [14], [15] and several mechanisms for their bactericidal effects have been proposed. Although only a few studies have reported the antibacterial properties of copper nanoparticles, they show copper nanoparticles have a significant promise as bactericidal agent [16]. However, other nanoparticles, such as platinum, gold, iron oxide, silica and its oxides, and nickel have not shown bactericidal effects in studies with Escherichia coli [15], [17], [18]. Yoon et al. [19] reported the antibacterial effects of silver and copper nanoparticles using single representative strains of E. coli and Bacillus subtilis, where the copper nanoparticles demonstrated superior antibacterial activity compared to the silver nanoparticles. Silver and copper nanoparticles supported on various suitable materials, such as carbon, polyurethane foam, polymers and sepiolite have also been effectively used for bactericidal applications [13], [14], [20], [21], [22]. While various hypotheses have been proposed to explain the mechanism of antimicrobial activity of silver nanoparticles, it is widely believed that silver nanoparticles are incorporated in the cell membrane, which causes leakage of intracellular substances and eventually causes cell death [12], [15]. Some of the silver nanoparticles also penetrate into the cells. It is reported that the bactericidal effect of silver nanoparticles decreases as the size increases and is also affected by the shape of the particles [23], [24]. Although most studies have utilized spherical particles, truncated triangular shaped particles are reported to have greater bactericidal effect compared to that of spherical and rod-shaped particles [24]. It is also reported that bactericidal efficiency is affected by the type of microorganism. In studies with gram negative, E. coli, and gram positive, Staphylococcus aureus, Kim et al. [2] reported greater biocidal efficiency of silver nanoparticles for E. coli, and attributed it to difference in cell wall structure between gram negative and gram positive microorganisms. However, currently there is insufficient evidence to support such conclusions since most research on bactericidal effect of nanoparticles has been conducted with one or a very limited number of microbial strains [12], [13], [14], [15].

The objective of this study was to compare the bactericidal effect of silver and copper nanoparticles using various microbial strains. Such a comparative study would reveal strain specificities and would eventually lead to better utilization of nanoparticles for specific application. Three representative bacteria typically recommended for use in antimicrobial assays, i.e., E. coli, B. subtilis and S. aureus were used and studies were conducted with eight strains, i.e. four E. coli strains, one B. subtilis strain and three S. aureus strains. The antimicrobial effect was quantified based on the inhibition zone measured in the disk diffusion tests conducted in plates and by determining the minimum growth inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) of nanoparticles in liquid batch cultures.

Section snippets

Materials and bacterial strains

The bactericidal experiments were carried out with gram negative bacteria E. coli and gram positive bacteria B. subtilis and S. aureus in nutrient media, composed of peptone (Loba Chemie Ltd., Mumbai) and NaCl (Merck Ltd., Mumbai) 5 g l−1 each, and yeast extract (Central Drug House, New Delhi) and beef extract (S.D. Fine Chem Ltd., Mumbai) 1.5 g l−1 each. Throughout this study, the same nutrient media was used for all strains, unless otherwise specified. For preparing solid media, the nutrient

Results and discussion

The EDS profile of silver nanoparticles (Fig. 1a) indicates that the sample contains pure silver, with no oxide layer. In contrast, an oxygen peak is observed in the EDS profile of the copper nanoparticles (Fig. 1b), suggesting the presence of an oxide layer. The XRD pattern of silver and copper nanoparticles (Fig. 2a and b) were compared and interpreted with standard data of International Centre of Diffraction Data (ICDD). The eight characteristic peaks for silver nanoparticles appeared at

Conclusions

Growth studies of different microbial cultures were performed in the presence of nanoparticles to observe their effect on the growth profile. This study shows that silver and copper nanoparticles have great promise as antimicrobial agent against E. coli, B. subtilis and S. aureus. MIC, MBC and disk diffusion test suggest that for all cultures of E. coli and S. aureus, the antimicrobial action of the silver nanoparticles were superior. Although an oxide layer was formed on the copper

Acknowledgements

The authors gratefully acknowledge the National Doctoral Fellowship (NDF) awarded by All India Council for Technical Education (AICTE), New Delhi, India, which provided funds for student support. The authors would like to acknowledge Department of Metallurgical Engineering and Material Science, IIT Bombay, for the XRD analysis, SAIF (sophisticated analytical instrument facility) IIT Bombay, for the EDS, ICP-AES and TEM analysis, and CRNTS (Centre for Research in Nanotechnology and Science) IIT

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