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Applied and Environmental Microbiology, August 2008, p. 5228-5230, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00086-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Effect of Silver-Doped Phosphate-Based Glasses on Bacterial Biofilm Growth{triangledown}

Sabeel P. Valappil,1,2 Jonathan C. Knowles,1 and Michael Wilson2*

Division of Biomaterials and Tissue Engineering,1 Division of Microbial Diseases, UCL Eastman Dental Institute, University College London, 256 Gray's Inn Road, London WC1X 8LD, United Kingdom2

Received 11 January 2008/ Accepted 14 June 2008


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ABSTRACT
 
Silver-containing phosphate-based glasses were found to reduce the growth of Pseudomonas aeruginosa and Staphylococcus aureus biofilms, which are leading causes of nosocomial infections. The rates of glass degradation (1.27 to 1.41 µg·mm–2·h–1) and the corresponding silver release were found to account for the variation in biofilm growth inhibitory effect.


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INTRODUCTION
 
Many of the hospital-acquired infections caused by Pseudomonas aeruginosa and Staphylococcus aureus (5, 7-9, 11, 17) are associated with biofilms and result in significant morbidity and mortality. Biofilm-associated bacteria show decreased susceptibilities to antibiotics (4), disinfectants (13), and clearance by host defenses (7, 16). Hence, the development of alternative strategies for combating biofilm-associated infections, such as the use of controlled metal ion-releasing phosphate-based glasses (PBGs), is of considerable interest (10, 19). Silver cations exhibit broad antimicrobial activity at low concentrations, and they are already being used for the treatment of burn wounds (14) and traumatic injuries (3). Ahmed et al. (1, 2) have shown that PBGs containing silver in its +1 oxidation state exert antibacterial effects against planktonic P. aeruginosa and S. aureus. Valappil et al. (19) reported that 10, 15, and 20 mol% levels of silver in PBGs were useful in preventing the growth of S. aureus biofilms. However, striking a balance between antimicrobial and cyto-/biocompatibility is of major importance for the in vivo clinical application of these PBGs. Therefore, the aim of this study was to prepare low-concentration (3 and 5 mol% silver) silver-doped PBGs and evaluate their efficacy against biofilms of P. aeruginosa and S. aureus.

PBGs for this study were produced using NaH2PO4 (BDH), P2O5 (Sigma), and CaCO3 (BDH) as described previously (19), and samples with the composition (P2O5)50(CaO)30(Na2O)20 (denoted Ag0) without silver were also prepared. Ag2SO4 (BDH) was also used for the preparation of silver-doped PBGs with the general composition (P2O5)50(CaO)30(Na2O)20 – x (Ag2SO4)x, where x is 3 or 5, hereafter given the abbreviations Ag3 and Ag5, respectively. Degradation was studied by measuring weight loss, and ion release was monitored using ion chromatography. Similarly, Ag+ release was measured using a test kit (Merck, United Kingdom), as described in detail elsewhere (19). The degradation rates, obtained by applying a line of best fit through the plot of weight loss per unit area of each glass against time (data not shown), for the Ag0, Ag3, and Ag5 glasses were 1.41, 1.27, and 0.83 µg·mm–2·h–1, respectively. The degradation profiles of the glasses showed decreases in degradation rate with increases in silver content (Fig. 1). As expected, the highest levels of Ca2+ and Na+ release were observed for the composition with the highest dissolution rate, Ag0. Ag+, Na+, and Ca2+ release profiles showed decreases with increasing silver content from Ag3 to Ag5 (Fig. 1). Among the anions (PO43–, P2O74–, P3O93–, and P3O105–), P3O93– was released to the greatest extent, but there were no significant differences between Ag3 and Ag5 (Fig. 1).


Figure 1
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  		FIG. 1. Relationship between cation and anion release rates and rates of degradation of silver-doped PBGs as a function of silver content.

Biofilm growth and inhibition studies were performed with a constant-depth film fermentor (University College Cardiff, Cardiff, United Kingdom), as described previously (12). Viable counts (CFU) were carried out as described previously (19), with MacConkey and nutrient agar plates used to grow P. aeruginosa and S. aureus, respectively. Statistical analyses of the data and t tests were conducted using GraphPad software (San Diego, CA). The initial surface attachments of P. aeruginosa and S. aureus on silver-doped PBGs were analyzed using scanning electron microscopy as described previously (19), and the attachments of S. aureus and P. aeruginosa biofilms to Ag3 and Ag5 were reduced compared to the levels for Ag0 and hydroxyapatite (HA) discs (data not shown). These results emphasize the need to explore the antiadhesive properties of silver, which could widen the applications of silver-containing antibacterial formulations, e.g., coating of catheters with silver ions to avoid bloodstream infections (6, 18). Confocal laser scanning microscopic analyses were also conducted, and the results showed that the numbers of nonviable bacteria in biofilms were higher for Ag3 and Ag5 glasses than for Ag0 glasses (data not shown).

P. aeruginosa biofilms on the Ag3 glasses showed significant differences (P = 0.007) in log10 viable count at 6 h compared to the levels for the controls (P = 0.012) (Fig. 2A), which became more apparent at 12 h. At 24 h, the log10 mean number of viable cells on the Ag3 glasses started to recover from the previous low at 12 h but was still less than the levels for both controls (P ≤ 0.001). At 48 h and 120 h, Ag3 continued to show fewer CFU than the controls (P ≤ 0.01). The Ag5 glasses showed no significant difference in log10 viable count compared to Ag0 (P = 0.12) or HA (P = 0.09) at 6 h (Fig. 2B), but there were significant differences (P ≤ 0.0001) at 12 h. At 24 h, the log10 viable count on the Ag5 glasses started to recover from the previous low at 12 h but was still less than the levels for both controls (P ≤ 0.0002). This effect was continued at the 48- and 120-h time points since Ag5 glasses showed fewer CFU than the controls (P ≤ 0.025).


Figure 2
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  		FIG. 2. (A) Log10 numbers of CFU/mm2 of P. aeruginosa in biofilms formed on HA, Ag0, and Ag3. (B) Log10 numbers of CFU/mm2 of P. aeruginosa in biofilms formed on HA, Ag0, and Ag5 glasses.

S. aureus biofilms on Ag3 glasses showed a significant difference in log10 viable count compared to the levels for the Ag0 glasses (P ≤ 0.022) but not to those for HA (P = 0.0965) at 6 h (Fig. 3A). At 24 h, the differences in log10 viable count increased compared to the levels for both controls, Ag0 (P = 0.0001) and HA (P = 0.0003). However, at 48 h, the log10 viable count on the Ag3 glasses started to recover from the previous low at 24 h but was still less than the levels for both controls (P ≤ 0.0002). At the 120- and 144-h time points, the Ag3 glasses continued to show fewer CFU than the Ag0 glasses (P ≤ 0.038) but not the HA discs (P ≤ 0.0973). The Ag5 glasses showed no significant difference in log10 viable count compared to Ag0 or HA discs (P ≤ 0.492) at 6 h (Fig. 3B), but the differences became apparent at 24 h (P ≤ 0.0001). At 48 h, the log10 viable count on the Ag5 glasses started to recover from the previous low at 24 h but was still less than the levels for both controls (P ≤ 0.0015). This effect was maintained until 144 h, as the Ag5 glasses continued to maintain CFU reductions of approximately 1.15 log10 compared to Ag0 and HA (P ≤ 0.014).


Figure 3
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  		FIG. 3. (A) Log10 numbers of CFU/mm2 of S. aureus in biofilms formed on HA, Ag0, and Ag3. (B) Log10 numbers of CFU/mm2 of S. aureus in biofilms formed on HA, Ag0, and Ag5 glasses.

The Ag3 and Ag5 glasses were more effective at reducing S. aureus biofilm growth at 24 h (1.73- and 2.10-µg·mm–2·h–1 reductions in log10 viable count, respectively, compared to the levels for silver-free glasses) than the Ag20 glass (1.34-µg·mm–2·h–1 reduction in log10 viable count compared to the levels for silver-free glasses), as reported previously (19). These effects might be due to the higher rates of glass degradation (1.27 and 1.41 µg·mm–2·h–1, respectively, for Ag3 and Ag5) and subsequent silver release (0.116 and 0.188 ppm h–1 for Ag3 and Ag5, respectively) achieved for these glasses compared to the levels for Ag20 (glass degradation rate of 0.42 µg·mm–2·h–1 and silver release rate of 0.064 ppm h–1), as reported previously (19). Thus, both Ag3 and Ag5 release sufficient quantities of Ag+ to reduce the growth of P. aeruginosa and S. aureus biofilms but are well within the acceptable cyto-/biocompatible range. It has been reported that the minimum biofilm growth inhibitory concentration of silver is 0.1 ppm, and the cytotoxic concentration is 1.6 ppm for human cells (15). The ion release profile confirmed that the Ag+ release was within the limits specified above, 0.116 ppm h–1 for the Ag3 compositions and 0.188 ppm h–1 for the Ag5 compositions. This study on the ability of Ag+ to reduce bacterial adhesion and to reduce biofilm growth is important for devising novel and efficient strategies for combating infections caused by P. aeruginosa and S. aureus biofilms.


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ACKNOWLEDGMENTS
 
This work was supported by the EPSRC, United Kingdom, grant no. GR/T21080/01.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Microbial Diseases, UCL Eastman Dental Institute, 256 Gray's Inn Road, London WC1X 8LD, United Kingdom. Phone: 44 (0)207 915 1050. Fax: 44 (0)207 915 1127. E-mail: M.Wilson{at}eastman.ucl.ac.uk Back

{triangledown} Published ahead of print on 20 June 2008. Back


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Applied and Environmental Microbiology, August 2008, p. 5228-5230, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00086-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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