Antibacterial effect of silver nanoparticles against

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Materials Letters 63 (2009) 2603–2606

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Materials Letters j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m a t l e t

Antibacterial effect of silver nanoparticles against Streptococcus mutans L.F. Espinosa-Cristóbal a, G.A. Martínez-Castañón a,⁎, R.E. Martínez-Martínez a, J.P. Loyola-Rodríguez a, N. Patiño-Marín a, J.F. Reyes-Macías a, Facundo Ruiz b a b

Maestria en Ciencias Odontológicas, Facultad de Estomatología, UASLP, Av. Manuel Nava 2, Zona Universitaria, San Luis Potosí, S. L. P., Mexico Facultad de Ciencias, UASLP, Álvaro Obregón 64, Col. Centro, San Luis Potosí, S. L. P., Mexico

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Article history: Received 12 August 2009 Accepted 6 September 2009 Available online 12 September 2009 Keywords: Antibacterial effect Streptococcus mutans Silver nanoparticles Caries

a b s t r a c t Dental caries is a very infectious disease; in humans, 95% of the worldwide population is affected. The microorganism associated with dental caries is Streptococcus mutans (S. mutans). Although several mechanisms for its control have been used, its prevalence and incidence are still high. New alternatives are silver nanoparticles due to their antibacterial effect. In this work, we determined the antibacterial effect of silver nanoparticles on S. mutans. Three sizes of silver nanoparticles were used to find minimum inhibitory concentrations (MIC) in S. mutans using reference and clinical stocks. Kruskal–Wallis and U of Mann–Whitney statistical tests were applied. We found bactericidal effect for the three groups, with significant statistical differences between them. Our results agree with those already reported by several authors. This study concludes that silver nanoparticles present antibacterial activity on S. mutans and this property is better when the particle size is diminished. © 2009 Elsevier B.V. All rights reserved.

1. Introduction

2. Materials and methods

Dental caries remains as one of the most widespread diseases of mankind, about 95% of the world population is affected in different ages of their lives [1–3] and, in both, developed and poor countries dental caries is still considered a public health problem [4]. Since the discovery of the Streptococcus mutans (S. mutans) as the etiologic agent of dental caries, the attention has been focused in this bacterium as a target in the prevention of the disease through the use of antimicrobial agents and the elaboration of a vaccine [5,6]. The use of some antimicrobials and the addition of fluoride to several dietetic supplements have resulted in the diminution of the prevalence of dental caries [1], but it has been reported that antibiotics and chemical bactericides often disturb the bacterial flora of the oral cavity and digestive tract and the development of multidrug-resistant strains of bacteria is also possible [6–9]; also, in some cases the use of fluoride is not very effective [10]. For this reason, there is a need in the use of an agent who does not generate resistance and presents a good bactericidal property. Silver nanoparticles have a great bactericidal effect on a several range of microorganisms, its bactericidal effect is known very well and the bactericidal effect of nanoparticles depends on the size and the shape of the particle [12–15]. In this work, we used silver nanoparticles of three different sizes on S. mutans to know the inhibition activity of the silver nanoparticles using clinical stocks and reference bacteria of S. mutans. Until our knowledge there are not reports on the bactericide activity of silver nanoparticles against S. mutans from clinical stocks.

2.1. Synthesis of silver nanoparticles

⁎ Corresponding author. Tel./fax: +52 444 8262361. E-mail address: [email protected] (G.A. Martínez-Castañón). 0167-577X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.matlet.2009.09.018

Silver nanoparticles with three different sizes were prepared following a method which is a modification of that reported by Martínez-Castañón et al. [11]. All preparations started with a 0.01 M AgNO3 solution placed in a 250 mL reaction vessel. Under magnetic stirring, 10 mL of deionized water containing gallic acid was added to the Ag+ solution. After the addition of gallic acid the pH value of the solution was immediately adjusted (for sample A the pH was raised to 11 with NaOH 1.0 M and for sample B pH was raised to 10 with NH4OH). For sample C, after the addition of gallic acid the mixture was irradiated with UV light (254 nm, 15 W) during 30 min (pH was not modified). After this time, the solution was heated during 30 min at 80 ºC. 2.2. Characterization of silver nanoparticles The obtained silver nanoparticles were characterized using Dynamic Light Scattering in a Malvern Zetasizer Nano ZS. Transmission Electron Microscopy (TEM) analysis was performed on a JEOL JEM-1230 at an accelerating voltage of 100 kV. 2.3. Antibacterial test The applied antibacterial test was reported by Loyola-Rodríguez et al. [16]. The S. mutans strains used in this study were 30 clinical stocks (associated with active caries; identified and classified by P.C.R. [Polymerase Chain Reaction]) and one reference stock (ATCC® 25175™). These strains were cultured in brain–heart–broth infusion

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by 18 h at 37 °C before the test. Microdilution plates were used, 200 μL of the dispersion form of silver nanoparticles was placed in the first column and then it was diluted 1:1 with brain–heart infusion with 2%

sucrose inoculated with S. mutans at 6 × 105 CFU/mL; the plates were incubated at 37 °C for 24 h. After incubation, all wells of plates were washed and then fixed with glutaraldehyde; after that, they were

Fig. 1. TEM and DLS results of the silver nanoparticles prepared in this work.

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Fig. 1 (continued).

washed and colored with 0.5% crystal violet. The minimum inhibitory concentration (MIC) was read in the last well that presented coloration. The nanoparticle dispersions were used in the form in which they had been prepared. The tests were made in triplicate. The values were analyzed with Kruskal–Wallis and U-Mann–Whitney tests. 2.4. Atomic Force Microscopy (AFM) analysis S. mutans cells were observed using AFM (Nanosurf Easyscan 2) before and after the treatment with silver nanoparticles. To do this, 30 µL of BHI broth containing bacteria (treated or not treated) was passed through a cellulose membrane filter (0.2 μm pore diameter), the filter was dried at room temperature during 5 days and after that, AFM images were collected in Contact Mode using a SiN tip with a 450 × 50 μm2 cantilever, with a force constant and a resonant frequency of 13 kHz and 0.2 N/m respectively and at 256 points/line and 1 s/line. 3. Results and discussion

3.2. Antibacterial test The results of the antibacterial test are presented as average values in Table 1. The three different silver nanoparticles are active bactericidal agents and the antimicrobial activities depend on their sizes, with a statistical significant difference when comparing the three groups. The nanoparticles with the lower MIC were the 8.4 nm nanoparticles followed by the 16.1 and 98 nm nanoparticles in that order, agreeing with results reported for E. coli and S. aureus [11,14]. For the three sizes, the MIC found was lower when the particles were tested against the standard stock, these results agree with those reported by Yoo et al. They reported differences in the susceptibility between standard strains and clinical isolates [17]. Hernandez-Sierra et al. worked with several kinds of nanoparticles which included silver nanoparticles of 25 nm. They used a reference stock of S. mutans and obtained a minimal inhibitory concentration of 4.86 µg/mL [18]. This result is lower than that obtained in this work and could be due to the method used. We used a method that includes the addition of sucrose. This enhances the cariogenic power of S. mutans and gives us a more realistic result because this compound is always present in our diet.

3.1. Characterization of silver nanoparticles Using X-ray diffraction analysis (results not shown) the nanoparticles were identified as elemental silver. Fig. 1 shows TEM images and the results of DLS analysis. The nanoparticles prepared have a narrow size distribution and present spherical and pseudospherical shape. In DLS analyses (insets in Fig. 1a, b and c) silver nanoparticles present peaks centered at 8.4, 16.1 and 98 nm for samples A, B and C respectively. These results confirm a good stabilization of the nanoparticles by gallic acid. Since the morphology of the obtained nanoparticles does not change significantly with size we can attribute the differences in the antibacterial results only to the differences in size.

Table 1 Minimum inhibitory concentration of silver nanoparticles against 30 clinical and 1 reference stocks of Streptococcus mutans. Sample (size, nm)

Clinical isolates Average ± SD (µg/mL)a

Standard reference Average ± SD (µg/mL)b

A (8.4) B (16.1) C (98)

101.98 ± 72.03 145.64 ± 104.88 320.63 ± 172.83

66.87 ± 0 108.33 ± 77.22 222.92 ± 77.22

a b

Average results for 30 clinical stocks in triplicate. Average results for one reference stock in triplicate.

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Fig. 2. AFM images of S. mutans, a) without treatment; b) treated with 8.4 nm silver nanoparticles; c) treated with 16.1 nm silver nanoparticles and d) treated with 98 nm silver nanoparticles.

The morphology of S. mutans before and after their treatment with silver nanoparticles was observed using AFM (Fig. 2). We can observe that cell membrane did not change for any of the three sizes of silver nanoparticles; we can say that silver nanoparticles present the same mechanism despite their size, the difference comes from their superficial area. The smaller the nanoparticle the more it releases Ag+ ions and their antibacterial effect can be better. Due to the limits of the microscope silver nanoparticles cannot be seen in Fig. 2. 4. Conclusions Silver nanoparticles of three different sizes were prepared and characterized, it was found that they present antibacterial activity against S. mutans and this property depends on the size of the particles. Until our knowledge this is the first report that tests the antibacterial property of silver nanoparticles on clinical stocks of S. mutans. Acknowledgements This work was partially supported by Consejo Nacional de Ciencia y Tecnología (CONACYT, Grant 80401), Programa de Mejoramiento del Profesorado (PROMEP, Grant PROMEP/103.5/08/5521) and Fondo de Apoyo a la Investigación (FAI, Grant C08-FAI-10-3.39) of the Universidad Autónoma de San Luis Potosí (UASLP).

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