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Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis
Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis Kalimuthu Kalishwaralal, Selvaraj BarathManiKanth, Sureshbabu Ram Kumar Pandian, Venkataraman Deepak, Sangiliyandi Gurunathan ∗ Department of Biotechnology, Division of Molecular and Cellular Biology, Kalasalingam University, Anand Nagar, Krishnankoil 626190, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 7 January 2010 Received in revised form 14 April 2010 Accepted 15 April 2010 Available online 22 April 2010 Keywords: Anti-biofilm activity Silver nanoparticles Keratitis Pseudomonas aeruginosa Staphylococcus epidermidis
a b s t r a c t Biofilms are ensued due to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix. Formation of these sessile communities and their inherent resistance to anti-microbial agents are the source of many relentless and chronic bacterial infections. Such biofilms are responsible play a major role in development of ocular related infectious diseases in human namely microbial keratitis. Different approaches have been used for preventing biofilm related infections in health care settings. Many of these methods have their own demerits that include chemical based complications; emergent antibiotic resistant strains, etc. silver nanoparticles are renowned for their influential anti-microbial activity. Hence the present study over the biologically synthesized silver nanoparticles, exhibited a potential anti-biofilm activity that was tested in vitro on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epidermidis during 24-h treatment. Treating these organisms with silver nanoparticles resulted in more than 95% inhibition in biofilm formation. The inhibition was known to be invariable of the species tested. As a result this study demonstrates the futuristic application of silver nanoparticles in treating microbial keratitis based on its potential anti-biofilm activity. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Contact lenses (CL) often get infected with bacteria, and prolonged usage of such lenses leads to microbial keratitis in eye. Bacteria frequently adhere to the surface of the lens through a biofilm matrix, a three-dimensional, gel-like, highly hydrated and locally charged environment, and using such lens can cause infections in eye. Adhesion of these bacteria to CL may contribute to the pathogenesis of infection and may be influenced by lens surface properties [1–3]. The first step in biofilm formation is the adhesion of microbial cell to the surface by the exopolysaccharides synthesized by the bacteria [4]. Pseudomonas aeruginosa and Staphylococcus epidermidis have been well-known as the major causative agents of infectious keratitis. The Gram-negative opportunistic aerobic rod P. aeruginosa is ubiquitous and well adapted for growth in an aquatic environment, also synthesizes an adhesive alginate extracellular matrix for biofilm formation. The Gram-positive aerobic coccus S. epidermidis is present as normal saprophytic flora on the skin, and it produces a surface polysac-
∗ Corresponding author at: Department of Biotechnology & Chemical Engineering, Division of Molecular and Cellular Biology, Kalasalingam University (Kalasalingam Academy of Research and Education), Anand Nagar, Krishnankoil 626 190, Tamilnadu, India. Tel.: +91 4563 289042; fax: +91 4563 289322. E-mail address: [email protected] (S. Gurunathan). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.04.014
charide that is involved in adherence and biofilm formation. These two organisms are capable of adhesion and biofilm formation on CL and even on the inner walls of lens storage cases [5–10]. The cells in the biofilm are distinct from their planktonic counterparts, since the biofilm augments resistance to drug therapy, disinfectants, and the immune response of the host [7–9]. The biofilm on CL prolongs the bacteria’s contact with the surface of the eye, thus increasing its pathogenicity [10]. Earlier, in the 19th century, microbial infections were treated with 0.5% AgNO3 like Ophthalmia neonatorum (by German obstetrician Carl Crede), and for the prevention of infection in burns. When the era of the antibiotics began with the discovery of penicillin, the use of silver slowly diminished [11]. But in the present scenario due to the emergence of biocide-resistant strains, once again the use of silver for treating infections has gained importance. However, the use of ionic silver has one major drawback; they are easily inactivated by complexation and precipitation thus limiting the uses [12]. Here zerovalent silver nanoparticles can be a valuable alternative for ionic silver [13]. Nanosilver is one of non-toxic and safe antibacterial agents to the human body. Besides, silver nanoparticles are also reported to possess anti-fungal activity [14], anti-inflammatory effect [15], anti-viral activity [16] and anti-angiogenic activity [17,18]. But, silver nanoparticles can be well applied in therapy safely when the effective concentrations of silver nanoparticles on various types of organisms are determined.
K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344
There are many methods available for the synthesis of silver nanoparticles. Most chemical methods use a reducing agent (e.g, sodium borohydride) to reduce Ag+ to Ag0 and a stabilizer (e.g., polyvinylpyrrolidone) to control particle growth and prevent aggregation. However, these preparations often have problems with particle stability and are difficult to scale up. In addition, there is a demand for more environment-friendly production methods. Alternatively, silver nanoparticles can also be synthesized biologically using bacteria [13]. The present study divulges the anti-microbial and anti-biofilm ability of biologically synthesized silver nanoparticles against P. aeruginosa and S. epidermidis, the important causative agents of keratitis. To our knowledge, this is the first report on the antibiotic effect of silver nanoparticles on P. aeruginosa and S. epidermidis and its effect on the biofilm formation. 2. Materials and methods 2.1. Strains used
341
Therefore assuming 100% conversion of all silver ions to silver nanoparticles, 3
N=
3.14 × 10.5 × (50.0 × 10−7 ) × 6.023 × 1023 6 × 107.868
i.e. N = 3837233.003 • Determine the molar concentration of the nanoparticle solution using the following formula: (Liu et al. [20]) C=
NT NVNA
where C is the molar concentration of nanoparticle solution, NT is the total number of silver atoms added as AgNO3 = 1 M, N is the number of atoms per nanoparticle (from calculation 1), V is the volume of the reaction solution in L, NA is the Avogadro’s number (=6.023 × 1023 ).
Wild-type of Bacillus licheniformis, P. aeruginosa and S. epidermidis were maintained in nutrient agar as well as sub cultured from time to time in the microbiology laboratory during the study period.
C=
2.2. Synthesis of silver nanoparticles
C = 2.606 × 10−7 M/L = 260 nM Further, the required concentration are made out from the obtained values.
In a typical experiment, 2 g of wet B. licheniformis biomass was taken in an Erlenmeyer’s flask. 1 mM AgNO3 solution was prepared using deionized water and 100 mL of the solution mixture was added to the biomass. Then the conical flask was kept in a shaker at 37 ◦ C (200 rpm) for 24 h for the synthesis of nanoparticles [19]. 2.3. Purification of silver nanoparticles The cells from each Erlenmeyer flask were washed twice with 50 mM phosphate buffer (pH 7.0) and re-suspended in 5 mL of the same buffer. Ultrasonic disruption of cells was carried out with an ultrasonic processor (Sonics Vibra Cell VC-505/220, Newtown, USA) over three 15 s periods, and with an interval of 45 s between periods. The resulting solution was centrifuged (16,000 rpm, 30 min) and filtered through a 0.22 m filter (Millipore,) to remove celldebris. Characterization of synthesized and purified particles was carried out according to the method described previously [19]. Samples for transmission electron microscopy (TEM) analysis were prepared on carbon-coated copper TEM grids. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage of 120 kV. 2.4. Determination of concentration of the silver nanoparticles The concentration of silver nanoparticles was determined by the method which has been previously reported for Liu et al. [20] for gold nanoparticles. The calculation is as follows [20]. • To determine the average number of atoms per nanoparticle (Liu et al. [20]) N=
D3 NA 6M
where N is the number of atoms per nanoparticles, = 3.14, is the density of face centered, cubic (fcc) silver (=10.5 g/cm3 ), D is the average diameter of nanoparticles (=50 nm = 50 × 10−7 cm), M is the atomic mass of silver (=107.868 g), NA is the number of atoms per mole (Avogadro’s number) (=6.023 × 1023 ).
1 × 6.023 × 1023 3837233.003 × 1 × 6.023 × 1023
2.5. Determination of anti-microbial activity by well-diffusion method The AgNPs synthesized from B. licheniformis were tested for antimicrobial activity by conventional well-diffusion method against P. aeruginosa and S. epidermidis [21]. The pure cultures of organisms were sub cultured on nutrient broth at 37 ◦ C on a rotary shaker at 200 rpm. Wells of 6-mm diameter were made on Muller–Hinton agar plates using gel puncture. Each strain was spread uniformly onto the individual plates using sterile cotton swabs. Using a micropipette, 100 nM of the sample of nanoparticles solution was filled onto each well on all plates. After incubation at 37 ◦ C for 28 h, the different levels of zone of inhibition were measured. 2.6. Determination of biofilm formation by Congo red agar method (CRA) The determination of biofilm formation was carried out by the method described by Freeman et al. [22]. This method utilizes a specially prepared solid medium – brain heart infusion broth (BHI) supplemented with 5% sucrose and Congo red for screening the formation of biofilm by P. aeruginosa and S. epidermidis. The medium composes of BHI (37 g/L), sucrose (50 g/L), agar No.1 (10 g/L) and Congo red stain (0.8 g/L). Congo red was prepared in the form of concentrated aqueous solution and it was autoclaved at 121 ◦ C for 15 min, separately from other medium constituents. Following autoclave, the concentrated solution was added to agar which was previously cooled to 55 ◦ C. Plates were inoculated and incubated aerobically for 24–48 h at 37 ◦ C. 2.7. Tissue culture plate method (TCP) – in vitro biofilm formation assay To determine the efficacy of silver nanoparticles in elimination of formed biofilm, TCP method was carried out with suitable modifications [23]. Individual wells of sterile, polystyrene, 96-well-flat bottom tissue culture plates were filled with 180 L of BHI broth and inoculated with 10 L of overnight culture. To the mixture
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10 L of silver nanoparticles were added from the stock so that final concentration was made between 10 nM and 100 nM. The tissue culture plates were incubated for 24 h at 37 ◦ C. After incubation, content of each well was gently removed. The wells were washed four times with 0.2 mL of phosphate buffer saline (PBS pH 7.2) to remove free-floating ‘planktonic’ bacteria. Biofilms formed by adherent ‘sessile’ organisms in plate were fixed with sodium acetate (2%) and stained with crystal violet (0.1%, w/v). Excess stain was rinsed off by thorough washing with deionized water and plates were kept for drying. After drying, 95% ethanol was added to the wells and the optical densities (OD) of stained adherent bacteria were determined with a microplate reader (model 680, Bio-rad) at 595 nm (OD595 nm). These OD values were considered as an index of bacteria adhering to surface and forming biofilms. Experiment was performed in triplicate, the data was then averaged and the standard deviation was calculated. Fig. 1. TEM image obtained from purified AgNPs synthesized using B. licheniformis. Several fields were photographed and were used to determine the diameter of nanoparticles. The range of observed diameter was 50 ± 5 nm.
3. Results 3.1. Biological synthesis of AgNPs Silver nanoparticles were synthesized using B. licheniformis and purified. The synthesized nanoparticles were primarily characterized by UV–vis spectroscopy, which has proved to be a very useful technique for the analysis of nanoparticles [19]. Further characterization was carried out using transmission electron microscopy. Electron microscopic images show that purified nanoparticles are spherical with a mean diameter of 50 nm (Fig. 1). 3.2. Anti-microbial activity of AgNPs against P. aeruginosa and S. epidermidis AgNPs are one of the successfully employed anti-microbial agents commercially. Here, the anti-microbial activity of AgNPs was tested against P. aeruginosa and S. epidermidis, using well-diffusion method (Fig. 2). The zone of inhibition is found to be slightly higher for S. epidermidis in comparison to its counterpart. The zone of inhibition is given as a mean of four replicates of the diameter of inhibition zones (in mm) around each well with AgNPs solution (Table 1). 3.3. Detection of biofilm formation Biofilm formation is detected in many organisms synthesizing exopolysachharides. The biofilm is made up of microorganisms adhering to the surface coated with slime – the exopolysaccharide matrix which protects the microbes from the unfavorable
Table 1 Diameter of the zone of inhibition by silver nanoparticles against microbes responsible for the disease pathogenesis. Results are mean ± SD (n = 4). Microorganism
Diameter of inhibition zones (in mm) (mean of four replicates)
Staphylococcus epidermidis Pseudomonas aeruginosa
12 ± 1.2 mm 9.5 ± 0.9 mm
environmental factors [23]. Biofilm formation by P. aeruginosa and S. epidermidis were tested by growing the organism in Brain heart infusion agar supplemented with Congo red (BHIC) with and without silver nanoparticles. When the colonies were grown without AgNPs in the medium, the organisms appeared as dry crystalline black colonies, indicating the production of exopolysachharides, which is the prerequisite for the formation of biofilm (Fig. 3). Whereas when the organisms were grown on BHIC with AgNPs, the organisms did not survive. During the treatment with reduced concentrations of AgNPs (10 nM), the organisms continued to grow, but AgNPs treatment has inhibited the synthesis of exopolysachharides, indicated by the absence of dry crystalline black colonies (Fig. 3). The presence of nanoparticles at a certain level inhibited bacterial growth by more than 90%. When the exopolysachharide synthesis is arrested, the organism cannot form biofilm. Therefore, 50 nM of silver nanoparticles significantly arrested biofilm formation without affecting viability, whereas 100 nM inhibited the growth of the organism itself.
Fig. 2. Anti-microbial activity of the purified silver nanoparticles against P. aeruginosa (A) and S. epidermidis (B) at 100 nM concentration.
K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344
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Fig. 3. Ability of the organisms was checked for biofilm formation in brain heart infusion agar supplemented with Congo red. The appearance of black crystalline colonies indicate the exopolysachharide production by P. aeruginosa (A) and S. epidermidis (C), whereas the addition of 50 nM silver nanoparticles blocked the exopolysachharide synthesis by P. aeruginosa (B) and S. epidermidis (D).
3.4. Quantification of biofilm detachment As biofilm formation is a prerequisite for Microbial keratitis, the ability of P. aeruginosa and S. epidermidis, the major participitants, in the microbial biofilm formation was investigated in vitro by monitoring the binding of the dye crystal violet to adherent cells which directly revealed their effective ability in formation of biofilm [24]. Crystal violet staining, a spectrophotometrical method was adopted to quantify biofilm density, a technique used for quantification of biofilm [23]. A calibration curve was previously plotted using various concentration of crystal violet to arrive at the results. P. aeruginosa and S. epidermidis were grown to form biofilm for 24 h in microtiter plate wells and then treated with varying concentrations of silver nanoparticles (Fig. 4). Treatment for 2 h with concentration of 100 nM of silver nanoparticles resulted in a decrease of 95% and 98% of the biofilm formed and 50 nM resulted in a 50% reduction in biofilm. These data demonstrate that silver nanoparticles induced detachment of P. aeruginosa and S. epidermidis biofilms was rapid, efficient and also occurred at clinically achievable concentrations of silver nanoparticles. 4. Discussion Contact lenses associated MK is a severe eye infection arising from the presence of adhered microorganisms on the lens surface. MK is common among patients suffering from ocular injury [25], however other factors like continuous and overnight wear [25,26] contaminated lens care solutions and lens cases [27] are also involved in the development of MK. P. aeruginosa and Staphylococci sp. are the common causative pathogens of the MK. Therefore the ability of the nanoparticles to inhibit the formation of the biofilms and formed biofilms were checked against the aforesaid organisms. The exopolysaccharides synthesized by the bacteria
Fig. 4. The anti-biofilm ability checked by tissue culture plate method showing that addition of increasing concentrations of silver nanoparticles reduced the ability of the organisms to form biofilm and attach to the surface of the wells. 100 nM silver nanoparticles completely inhibited the exopolysachharide synthesis by both the orgamisms (silver nanoparticle treatment to the adhered (A) P. aeruginosa and (B) S. epidermidis on the wells).
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not only protects the bacteria from the host defense mechanism but also mediates the adhesion of the organism between the lens and the corneal epithelium [28,29]. Moreover the ability of the organisms to form biofilm was confirmed by the formation of dry crystalline colonies on BHIC. Here we checked the ability of the AgNPs to inhibit the growth of the organisms under consideration, by well-diffusion method of antibiotic assay. The organisms’ growth was effectively impeded by the silver nanoparticles at the concentration of 100 nM. The concentration of silver nanoparticles was lesser than the previous reports for the toxic concentration of the AgNPs in vitro against the mammalian retinal cells [18]. The recurrence of biofilms in spite of treatment with various anti-microbial agents was attributed to the impedance created by the biofilm matrix [30]. Although water channels are present in the biofilms, the deep lying organisms escape the treatment as the matrix hinders the diffusion of the drug. Therefore, inhibition of biofilm formation is very much essential in the case of prevention of MK and various other disorders, as the exopolysachharide slime formed reduces the susceptibility of the organism to the administered drug. Antibiotic treatments effectively kill the bacteria which remain individual, but the efficiency is very much reduced when the organism forms slime. This makes the organisms to revert the disease when the antibiotic treatment is finished because of the healing of symptoms. This is the reason behind the biofilm infections showing recurring symptoms even after cycles of antibiotic therapy until the mass is surgically removed. Therefore in this report we investigated whether AgNPs has anti-microbial activity and also anti-biofilm function by using BHIC. Here, AgNPs not only inhibited the growth but also the ability of the organism to synthesize the exopolysachharide. This is evident from Fig. 3 where the BHI agar containing Congo red is supplemented with reduced the concentration of AgNPs (50 nM). The concentration was chosen based on the non-inhibitory concentration values which only impeded the synthesis of the exopolysachharides but not the viability of the organism (data not shown). The organism continued to grow, but its ability to synthesize the exopolysachharide is blocked. This shows that silver nanoparticles have the ability to block the exopolysachharide synthesis of the organism otherwise the biofilm. Another method which was found to be useful in determining the ability of the moiety to disrupt the biofilm formation is the crystal violet method on TCP. The TCP assay described by Christensen et al. [23] is most widely used and was considered as standard test for detection of biofilm formation. The ability of the organism to form biofilm and the effect of various concentrations of silver nanoparticles in inhibiting the formation was checked on a 96-well plate, where an increase of silver nanoparticles concentration negatively regulated the biofilm formation. This is evident from Fig. 4 where 100 nM of silver nanoparticles treatment almost completely inhibited the formation of biofilm (>95%). Therefore the results obtained directly reveals that biologically synthesized silver nanoparticles at a concentration of 100 nM not only effectively inhibited the growth of the bacteria, but also wiped out the biofilm formed by it. This inhibitory effect of AgNPs on the existing biofilm may due to be presence of water channels though out the biofilm. Since in all biofilms water channels (pores) are present for nutrient transportation, AgNPs may directly diffuse through the exopolysaccharide layer through the pores and may impart anti-microbial function. The experiment with 96-well plate shows that AgNPs eliminate the biofilm formed previously besides inhibiting the formation of biofilm in existing bacteria.
5. Conclusion The present study characterizes the anti-biofilm activity of silver nanoparticles against two common bacterial pathogens that are been proven for their efficient biofilm formation. It is noteworthy that for prevention of Microbial keratitis, silver nanoparticles composing gel may be used as a safe biocide for destroying different bacterial biofilms. Observations made through microtiter plate assay (0.1% crystal violet staining) discloses the potential of silver nanoparticles in effective inhibition of biofilm formation which affirms the futuristic applications of AgNP based contact lens care solutions, for biofilm based human ocular problems, thereby serving mankind through an economic therapeutic alternative. Acknowledgements The authors gratefully acknowledge Dr. Pushpa Viswanathan, Professor, Cancer Institute (WIA), Chennai, India, for her immense support in analyzing samples under Transmission Electron Microscope. The author Kalishwaralal Kalimuthu is grateful to CSIR for providing senior research fellowship (Ack. No. 142070/2K9/1). References [1] J.P. Whitcher, M. Srinivasan, M.P. Upadhya, Bull. World Health Organ. 79 (2001) 214–221. [2] L. Kodjikian, E. Casoli-Bergeron, F. Malet, Arch. Clin. Exp. Ophthalmol. 246 (2008) 267–273. [3] E.N. Martins, M.E. Farah, L.S. Alvarenga, M.C. Yu, A.L. Höflin-Lima, CLAO J. 28 (2002) 146–148. [4] Y.N. Dang, A. Rao, P.R. Kastl, R.C. Blake, M.J. Schurr, D.A. Blake, Eye Contact Lens 29 (2003) 65–68. [5] V. Butcko, T.T. McMahon, C.E. Joslin, L. Jones, Eye Contact Lens 33 (2007) 421–425. [6] M. Henriques, C. Sousa, M. Lira, Optom. Vis. Sci. 82 (2005) 446–450. [7] J.W. Costerton, L. Montanaro, C.R. Arciola, Int. J. Artif. Organs 28 (2005) 1062–1068. [8] N. Hoiby, H. Krogh Johansen, C. Moser, Z. Song, O. Ciofu, A. Kharazmi, Microbes Infect. 3 (2001) 23–35. [9] K.K. Jefferson, FEMS Microbiol. Lett. 236 (2004) 163–173. [10] L. McLaughlin-Borlace, F. Stapleton, M. Matheson, J.K. Dart, J. Appl. Microbiol. 84 (1998) 827–838. [11] H.J. Klasen, Burns 26 (2000) 117–130. [12] B.S. Atiyeh, M. Costagliola, S.N. Hayek, S.A. Dibo, Burns 33 (2007) 139–148. [13] L. Sintubin, W.D. Windt, J. Dick, J. Mast, D. Vander Ha, W. Verstraete, N. Boon, Appl. Microbiol. Biotechnol. 84 (4) (2009) 741–749. [14] K.J. Kim, W.S. Sung, B.K. Suh, S.K. Moon, J.S. Choi, J.G. Kim, D.G. Lee, Biometals 22 (2009) 235–242. [15] P.L. Nadworny, J. Wang, E.E. Tredget, R.E. Burrell, Nanomedicine 4 (2008) 241–251. [16] J.V. Rogers Parkinson, Y.W. Choi, J.L. Speshock, S.M. Hussain, Nanoscale Res. Lett. 3 (2008) 129–133. [17] S. Gurunathan, K.J. Lee, K. Kalishwaralal, S. Sheikpranbabu, R. Vaidyanathan, S.H. Eom, Biomaterials 30 (2009) 6341–6350. [18] K. Kalishwaralal, E. Banumathi, S.R.K. Pandian, V. Deepak, J. Muniyandi, S.H. Eom, Colloid Surf. B: Biointerfaces 73 (2009) 51–57. [19] K. Kalimuthu, R.S. Babu, D. Venkataraman, M. Bilal, S. Gurunathan, Colloid Surf. B: Biointerfaces 65 (2008) 150–153. [20] X. Liu, M. Atwater, Q. Wang, J. Huo, Colloid Surf. B: Biointerfaces 58 (2007) 3–7. [21] A. Nanda, M. Saravanan, Nanomedicine 5 (2009) 452–456. [22] D.J. Freeman, F.R. Falkiner, C.T. Keane, J. Clin. Pathol. 42 (1989) 872–874. [23] G.D. Christensen, W.A. Simpson, J.A. Younger, L.M. Baddour, F.F. Barrett, D.M. Melton, J. Clin. Microbiol. 22 (1985) 996–1006. [24] H.M. Judith, E.K. Daniel, A.O. George, Curr. Protoc. Microbiol. (2005), 1B.1.1–1B.1.17. [25] K.H. Cheng, S.L. Leung, H.W. Hoekman, W.H. Beekhuis, P.G. Mulder, A.J. Geerards, Lancet 354 (1999) 181–185. [26] B.A. Weissman, B.J. Mondino, Contact Lens Anterior Eye 25 (2002) 3–9. [27] S.M.J. Fleiszig, D.J. Evans, Clin. Exp. Optom. 85 (2002) 271–278. [28] A.S. Hoffman, D. Schoen Mack, P. Becker, I. Chattergee, S. Dobinsky, J.K.M. Knobloch, G. Peters, Int. J. Med. Microbiol. 294 (2004) 203–212. [29] N. Nayak, T.C. Nag, G. Satpathy, S.B. Ray, Indian J. Med. Res. 125 (2007) 767–771. [30] A. Mohammed, L. Al-Fattani, J. Med. Microbiol. 55 (2006) 999–1008.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis Kalimuthu Kalishwaralal, Selvaraj BarathManiKanth, Sureshbabu Ram Kumar Pandian, Venkataraman Deepak, Sangiliyandi Gurunathan ∗ Department of Biotechnology, Division of Molecular and Cellular Biology, Kalasalingam University, Anand Nagar, Krishnankoil 626190, Tamilnadu, India
a r t i c l e
i n f o
Article history: Received 7 January 2010 Received in revised form 14 April 2010 Accepted 15 April 2010 Available online 22 April 2010 Keywords: Anti-biofilm activity Silver nanoparticles Keratitis Pseudomonas aeruginosa Staphylococcus epidermidis
a b s t r a c t Biofilms are ensued due to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix. Formation of these sessile communities and their inherent resistance to anti-microbial agents are the source of many relentless and chronic bacterial infections. Such biofilms are responsible play a major role in development of ocular related infectious diseases in human namely microbial keratitis. Different approaches have been used for preventing biofilm related infections in health care settings. Many of these methods have their own demerits that include chemical based complications; emergent antibiotic resistant strains, etc. silver nanoparticles are renowned for their influential anti-microbial activity. Hence the present study over the biologically synthesized silver nanoparticles, exhibited a potential anti-biofilm activity that was tested in vitro on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epidermidis during 24-h treatment. Treating these organisms with silver nanoparticles resulted in more than 95% inhibition in biofilm formation. The inhibition was known to be invariable of the species tested. As a result this study demonstrates the futuristic application of silver nanoparticles in treating microbial keratitis based on its potential anti-biofilm activity. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Contact lenses (CL) often get infected with bacteria, and prolonged usage of such lenses leads to microbial keratitis in eye. Bacteria frequently adhere to the surface of the lens through a biofilm matrix, a three-dimensional, gel-like, highly hydrated and locally charged environment, and using such lens can cause infections in eye. Adhesion of these bacteria to CL may contribute to the pathogenesis of infection and may be influenced by lens surface properties [1–3]. The first step in biofilm formation is the adhesion of microbial cell to the surface by the exopolysaccharides synthesized by the bacteria [4]. Pseudomonas aeruginosa and Staphylococcus epidermidis have been well-known as the major causative agents of infectious keratitis. The Gram-negative opportunistic aerobic rod P. aeruginosa is ubiquitous and well adapted for growth in an aquatic environment, also synthesizes an adhesive alginate extracellular matrix for biofilm formation. The Gram-positive aerobic coccus S. epidermidis is present as normal saprophytic flora on the skin, and it produces a surface polysac-
∗ Corresponding author at: Department of Biotechnology & Chemical Engineering, Division of Molecular and Cellular Biology, Kalasalingam University (Kalasalingam Academy of Research and Education), Anand Nagar, Krishnankoil 626 190, Tamilnadu, India. Tel.: +91 4563 289042; fax: +91 4563 289322. E-mail address: [email protected] (S. Gurunathan). 0927-7765/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.04.014
charide that is involved in adherence and biofilm formation. These two organisms are capable of adhesion and biofilm formation on CL and even on the inner walls of lens storage cases [5–10]. The cells in the biofilm are distinct from their planktonic counterparts, since the biofilm augments resistance to drug therapy, disinfectants, and the immune response of the host [7–9]. The biofilm on CL prolongs the bacteria’s contact with the surface of the eye, thus increasing its pathogenicity [10]. Earlier, in the 19th century, microbial infections were treated with 0.5% AgNO3 like Ophthalmia neonatorum (by German obstetrician Carl Crede), and for the prevention of infection in burns. When the era of the antibiotics began with the discovery of penicillin, the use of silver slowly diminished [11]. But in the present scenario due to the emergence of biocide-resistant strains, once again the use of silver for treating infections has gained importance. However, the use of ionic silver has one major drawback; they are easily inactivated by complexation and precipitation thus limiting the uses [12]. Here zerovalent silver nanoparticles can be a valuable alternative for ionic silver [13]. Nanosilver is one of non-toxic and safe antibacterial agents to the human body. Besides, silver nanoparticles are also reported to possess anti-fungal activity [14], anti-inflammatory effect [15], anti-viral activity [16] and anti-angiogenic activity [17,18]. But, silver nanoparticles can be well applied in therapy safely when the effective concentrations of silver nanoparticles on various types of organisms are determined.
K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340–344
There are many methods available for the synthesis of silver nanoparticles. Most chemical methods use a reducing agent (e.g, sodium borohydride) to reduce Ag+ to Ag0 and a stabilizer (e.g., polyvinylpyrrolidone) to control particle growth and prevent aggregation. However, these preparations often have problems with particle stability and are difficult to scale up. In addition, there is a demand for more environment-friendly production methods. Alternatively, silver nanoparticles can also be synthesized biologically using bacteria [13]. The present study divulges the anti-microbial and anti-biofilm ability of biologically synthesized silver nanoparticles against P. aeruginosa and S. epidermidis, the important causative agents of keratitis. To our knowledge, this is the first report on the antibiotic effect of silver nanoparticles on P. aeruginosa and S. epidermidis and its effect on the biofilm formation. 2. Materials and methods 2.1. Strains used
341
Therefore assuming 100% conversion of all silver ions to silver nanoparticles, 3
N=
3.14 × 10.5 × (50.0 × 10−7 ) × 6.023 × 1023 6 × 107.868
i.e. N = 3837233.003 • Determine the molar concentration of the nanoparticle solution using the following formula: (Liu et al. [20]) C=
NT NVNA
where C is the molar concentration of nanoparticle solution, NT is the total number of silver atoms added as AgNO3 = 1 M, N is the number of atoms per nanoparticle (from calculation 1), V is the volume of the reaction solution in L, NA is the Avogadro’s number (=6.023 × 1023 ).
Wild-type of Bacillus licheniformis, P. aeruginosa and S. epidermidis were maintained in nutrient agar as well as sub cultured from time to time in the microbiology laboratory during the study period.
C=
2.2. Synthesis of silver nanoparticles
C = 2.606 × 10−7 M/L = 260 nM Further, the required concentration are made out from the obtained values.
In a typical experiment, 2 g of wet B. licheniformis biomass was taken in an Erlenmeyer’s flask. 1 mM AgNO3 solution was prepared using deionized water and 100 mL of the solution mixture was added to the biomass. Then the conical flask was kept in a shaker at 37 ◦ C (200 rpm) for 24 h for the synthesis of nanoparticles [19]. 2.3. Purification of silver nanoparticles The cells from each Erlenmeyer flask were washed twice with 50 mM phosphate buffer (pH 7.0) and re-suspended in 5 mL of the same buffer. Ultrasonic disruption of cells was carried out with an ultrasonic processor (Sonics Vibra Cell VC-505/220, Newtown, USA) over three 15 s periods, and with an interval of 45 s between periods. The resulting solution was centrifuged (16,000 rpm, 30 min) and filtered through a 0.22 m filter (Millipore,) to remove celldebris. Characterization of synthesized and purified particles was carried out according to the method described previously [19]. Samples for transmission electron microscopy (TEM) analysis were prepared on carbon-coated copper TEM grids. TEM measurements were performed on a JEOL model 1200EX instrument operated at an accelerating voltage of 120 kV. 2.4. Determination of concentration of the silver nanoparticles The concentration of silver nanoparticles was determined by the method which has been previously reported for Liu et al. [20] for gold nanoparticles. The calculation is as follows [20]. • To determine the average number of atoms per nanoparticle (Liu et al. [20]) N=
D3 NA 6M
where N is the number of atoms per nanoparticles, = 3.14, is the density of face centered, cubic (fcc) silver (=10.5 g/cm3 ), D is the average diameter of nanoparticles (=50 nm = 50 × 10−7 cm), M is the atomic mass of silver (=107.868 g), NA is the number of atoms per mole (Avogadro’s number) (=6.023 × 1023 ).
1 × 6.023 × 1023 3837233.003 × 1 × 6.023 × 1023
2.5. Determination of anti-microbial activity by well-diffusion method The AgNPs synthesized from B. licheniformis were tested for antimicrobial activity by conventional well-diffusion method against P. aeruginosa and S. epidermidis [21]. The pure cultures of organisms were sub cultured on nutrient broth at 37 ◦ C on a rotary shaker at 200 rpm. Wells of 6-mm diameter were made on Muller–Hinton agar plates using gel puncture. Each strain was spread uniformly onto the individual plates using sterile cotton swabs. Using a micropipette, 100 nM of the sample of nanoparticles solution was filled onto each well on all plates. After incubation at 37 ◦ C for 28 h, the different levels of zone of inhibition were measured. 2.6. Determination of biofilm formation by Congo red agar method (CRA) The determination of biofilm formation was carried out by the method described by Freeman et al. [22]. This method utilizes a specially prepared solid medium – brain heart infusion broth (BHI) supplemented with 5% sucrose and Congo red for screening the formation of biofilm by P. aeruginosa and S. epidermidis. The medium composes of BHI (37 g/L), sucrose (50 g/L), agar No.1 (10 g/L) and Congo red stain (0.8 g/L). Congo red was prepared in the form of concentrated aqueous solution and it was autoclaved at 121 ◦ C for 15 min, separately from other medium constituents. Following autoclave, the concentrated solution was added to agar which was previously cooled to 55 ◦ C. Plates were inoculated and incubated aerobically for 24–48 h at 37 ◦ C. 2.7. Tissue culture plate method (TCP) – in vitro biofilm formation assay To determine the efficacy of silver nanoparticles in elimination of formed biofilm, TCP method was carried out with suitable modifications [23]. Individual wells of sterile, polystyrene, 96-well-flat bottom tissue culture plates were filled with 180 L of BHI broth and inoculated with 10 L of overnight culture. To the mixture
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10 L of silver nanoparticles were added from the stock so that final concentration was made between 10 nM and 100 nM. The tissue culture plates were incubated for 24 h at 37 ◦ C. After incubation, content of each well was gently removed. The wells were washed four times with 0.2 mL of phosphate buffer saline (PBS pH 7.2) to remove free-floating ‘planktonic’ bacteria. Biofilms formed by adherent ‘sessile’ organisms in plate were fixed with sodium acetate (2%) and stained with crystal violet (0.1%, w/v). Excess stain was rinsed off by thorough washing with deionized water and plates were kept for drying. After drying, 95% ethanol was added to the wells and the optical densities (OD) of stained adherent bacteria were determined with a microplate reader (model 680, Bio-rad) at 595 nm (OD595 nm). These OD values were considered as an index of bacteria adhering to surface and forming biofilms. Experiment was performed in triplicate, the data was then averaged and the standard deviation was calculated. Fig. 1. TEM image obtained from purified AgNPs synthesized using B. licheniformis. Several fields were photographed and were used to determine the diameter of nanoparticles. The range of observed diameter was 50 ± 5 nm.
3. Results 3.1. Biological synthesis of AgNPs Silver nanoparticles were synthesized using B. licheniformis and purified. The synthesized nanoparticles were primarily characterized by UV–vis spectroscopy, which has proved to be a very useful technique for the analysis of nanoparticles [19]. Further characterization was carried out using transmission electron microscopy. Electron microscopic images show that purified nanoparticles are spherical with a mean diameter of 50 nm (Fig. 1). 3.2. Anti-microbial activity of AgNPs against P. aeruginosa and S. epidermidis AgNPs are one of the successfully employed anti-microbial agents commercially. Here, the anti-microbial activity of AgNPs was tested against P. aeruginosa and S. epidermidis, using well-diffusion method (Fig. 2). The zone of inhibition is found to be slightly higher for S. epidermidis in comparison to its counterpart. The zone of inhibition is given as a mean of four replicates of the diameter of inhibition zones (in mm) around each well with AgNPs solution (Table 1). 3.3. Detection of biofilm formation Biofilm formation is detected in many organisms synthesizing exopolysachharides. The biofilm is made up of microorganisms adhering to the surface coated with slime – the exopolysaccharide matrix which protects the microbes from the unfavorable
Table 1 Diameter of the zone of inhibition by silver nanoparticles against microbes responsible for the disease pathogenesis. Results are mean ± SD (n = 4). Microorganism
Diameter of inhibition zones (in mm) (mean of four replicates)
Staphylococcus epidermidis Pseudomonas aeruginosa
12 ± 1.2 mm 9.5 ± 0.9 mm
environmental factors [23]. Biofilm formation by P. aeruginosa and S. epidermidis were tested by growing the organism in Brain heart infusion agar supplemented with Congo red (BHIC) with and without silver nanoparticles. When the colonies were grown without AgNPs in the medium, the organisms appeared as dry crystalline black colonies, indicating the production of exopolysachharides, which is the prerequisite for the formation of biofilm (Fig. 3). Whereas when the organisms were grown on BHIC with AgNPs, the organisms did not survive. During the treatment with reduced concentrations of AgNPs (10 nM), the organisms continued to grow, but AgNPs treatment has inhibited the synthesis of exopolysachharides, indicated by the absence of dry crystalline black colonies (Fig. 3). The presence of nanoparticles at a certain level inhibited bacterial growth by more than 90%. When the exopolysachharide synthesis is arrested, the organism cannot form biofilm. Therefore, 50 nM of silver nanoparticles significantly arrested biofilm formation without affecting viability, whereas 100 nM inhibited the growth of the organism itself.
Fig. 2. Anti-microbial activity of the purified silver nanoparticles against P. aeruginosa (A) and S. epidermidis (B) at 100 nM concentration.
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Fig. 3. Ability of the organisms was checked for biofilm formation in brain heart infusion agar supplemented with Congo red. The appearance of black crystalline colonies indicate the exopolysachharide production by P. aeruginosa (A) and S. epidermidis (C), whereas the addition of 50 nM silver nanoparticles blocked the exopolysachharide synthesis by P. aeruginosa (B) and S. epidermidis (D).
3.4. Quantification of biofilm detachment As biofilm formation is a prerequisite for Microbial keratitis, the ability of P. aeruginosa and S. epidermidis, the major participitants, in the microbial biofilm formation was investigated in vitro by monitoring the binding of the dye crystal violet to adherent cells which directly revealed their effective ability in formation of biofilm [24]. Crystal violet staining, a spectrophotometrical method was adopted to quantify biofilm density, a technique used for quantification of biofilm [23]. A calibration curve was previously plotted using various concentration of crystal violet to arrive at the results. P. aeruginosa and S. epidermidis were grown to form biofilm for 24 h in microtiter plate wells and then treated with varying concentrations of silver nanoparticles (Fig. 4). Treatment for 2 h with concentration of 100 nM of silver nanoparticles resulted in a decrease of 95% and 98% of the biofilm formed and 50 nM resulted in a 50% reduction in biofilm. These data demonstrate that silver nanoparticles induced detachment of P. aeruginosa and S. epidermidis biofilms was rapid, efficient and also occurred at clinically achievable concentrations of silver nanoparticles. 4. Discussion Contact lenses associated MK is a severe eye infection arising from the presence of adhered microorganisms on the lens surface. MK is common among patients suffering from ocular injury [25], however other factors like continuous and overnight wear [25,26] contaminated lens care solutions and lens cases [27] are also involved in the development of MK. P. aeruginosa and Staphylococci sp. are the common causative pathogens of the MK. Therefore the ability of the nanoparticles to inhibit the formation of the biofilms and formed biofilms were checked against the aforesaid organisms. The exopolysaccharides synthesized by the bacteria
Fig. 4. The anti-biofilm ability checked by tissue culture plate method showing that addition of increasing concentrations of silver nanoparticles reduced the ability of the organisms to form biofilm and attach to the surface of the wells. 100 nM silver nanoparticles completely inhibited the exopolysachharide synthesis by both the orgamisms (silver nanoparticle treatment to the adhered (A) P. aeruginosa and (B) S. epidermidis on the wells).
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not only protects the bacteria from the host defense mechanism but also mediates the adhesion of the organism between the lens and the corneal epithelium [28,29]. Moreover the ability of the organisms to form biofilm was confirmed by the formation of dry crystalline colonies on BHIC. Here we checked the ability of the AgNPs to inhibit the growth of the organisms under consideration, by well-diffusion method of antibiotic assay. The organisms’ growth was effectively impeded by the silver nanoparticles at the concentration of 100 nM. The concentration of silver nanoparticles was lesser than the previous reports for the toxic concentration of the AgNPs in vitro against the mammalian retinal cells [18]. The recurrence of biofilms in spite of treatment with various anti-microbial agents was attributed to the impedance created by the biofilm matrix [30]. Although water channels are present in the biofilms, the deep lying organisms escape the treatment as the matrix hinders the diffusion of the drug. Therefore, inhibition of biofilm formation is very much essential in the case of prevention of MK and various other disorders, as the exopolysachharide slime formed reduces the susceptibility of the organism to the administered drug. Antibiotic treatments effectively kill the bacteria which remain individual, but the efficiency is very much reduced when the organism forms slime. This makes the organisms to revert the disease when the antibiotic treatment is finished because of the healing of symptoms. This is the reason behind the biofilm infections showing recurring symptoms even after cycles of antibiotic therapy until the mass is surgically removed. Therefore in this report we investigated whether AgNPs has anti-microbial activity and also anti-biofilm function by using BHIC. Here, AgNPs not only inhibited the growth but also the ability of the organism to synthesize the exopolysachharide. This is evident from Fig. 3 where the BHI agar containing Congo red is supplemented with reduced the concentration of AgNPs (50 nM). The concentration was chosen based on the non-inhibitory concentration values which only impeded the synthesis of the exopolysachharides but not the viability of the organism (data not shown). The organism continued to grow, but its ability to synthesize the exopolysachharide is blocked. This shows that silver nanoparticles have the ability to block the exopolysachharide synthesis of the organism otherwise the biofilm. Another method which was found to be useful in determining the ability of the moiety to disrupt the biofilm formation is the crystal violet method on TCP. The TCP assay described by Christensen et al. [23] is most widely used and was considered as standard test for detection of biofilm formation. The ability of the organism to form biofilm and the effect of various concentrations of silver nanoparticles in inhibiting the formation was checked on a 96-well plate, where an increase of silver nanoparticles concentration negatively regulated the biofilm formation. This is evident from Fig. 4 where 100 nM of silver nanoparticles treatment almost completely inhibited the formation of biofilm (>95%). Therefore the results obtained directly reveals that biologically synthesized silver nanoparticles at a concentration of 100 nM not only effectively inhibited the growth of the bacteria, but also wiped out the biofilm formed by it. This inhibitory effect of AgNPs on the existing biofilm may due to be presence of water channels though out the biofilm. Since in all biofilms water channels (pores) are present for nutrient transportation, AgNPs may directly diffuse through the exopolysaccharide layer through the pores and may impart anti-microbial function. The experiment with 96-well plate shows that AgNPs eliminate the biofilm formed previously besides inhibiting the formation of biofilm in existing bacteria.
5. Conclusion The present study characterizes the anti-biofilm activity of silver nanoparticles against two common bacterial pathogens that are been proven for their efficient biofilm formation. It is noteworthy that for prevention of Microbial keratitis, silver nanoparticles composing gel may be used as a safe biocide for destroying different bacterial biofilms. Observations made through microtiter plate assay (0.1% crystal violet staining) discloses the potential of silver nanoparticles in effective inhibition of biofilm formation which affirms the futuristic applications of AgNP based contact lens care solutions, for biofilm based human ocular problems, thereby serving mankind through an economic therapeutic alternative. Acknowledgements The authors gratefully acknowledge Dr. Pushpa Viswanathan, Professor, Cancer Institute (WIA), Chennai, India, for her immense support in analyzing samples under Transmission Electron Microscope. The author Kalishwaralal Kalimuthu is grateful to CSIR for providing senior research fellowship (Ack. No. 142070/2K9/1). References [1] J.P. Whitcher, M. Srinivasan, M.P. Upadhya, Bull. World Health Organ. 79 (2001) 214–221. [2] L. Kodjikian, E. Casoli-Bergeron, F. Malet, Arch. Clin. Exp. Ophthalmol. 246 (2008) 267–273. [3] E.N. Martins, M.E. Farah, L.S. Alvarenga, M.C. Yu, A.L. Höflin-Lima, CLAO J. 28 (2002) 146–148. [4] Y.N. Dang, A. Rao, P.R. Kastl, R.C. Blake, M.J. Schurr, D.A. Blake, Eye Contact Lens 29 (2003) 65–68. [5] V. Butcko, T.T. McMahon, C.E. Joslin, L. Jones, Eye Contact Lens 33 (2007) 421–425. [6] M. Henriques, C. Sousa, M. Lira, Optom. Vis. Sci. 82 (2005) 446–450. [7] J.W. Costerton, L. Montanaro, C.R. Arciola, Int. J. Artif. Organs 28 (2005) 1062–1068. [8] N. Hoiby, H. Krogh Johansen, C. Moser, Z. Song, O. Ciofu, A. Kharazmi, Microbes Infect. 3 (2001) 23–35. [9] K.K. Jefferson, FEMS Microbiol. Lett. 236 (2004) 163–173. [10] L. McLaughlin-Borlace, F. Stapleton, M. Matheson, J.K. Dart, J. Appl. Microbiol. 84 (1998) 827–838. [11] H.J. Klasen, Burns 26 (2000) 117–130. [12] B.S. Atiyeh, M. Costagliola, S.N. Hayek, S.A. Dibo, Burns 33 (2007) 139–148. [13] L. Sintubin, W.D. Windt, J. Dick, J. Mast, D. Vander Ha, W. Verstraete, N. Boon, Appl. Microbiol. Biotechnol. 84 (4) (2009) 741–749. [14] K.J. Kim, W.S. Sung, B.K. Suh, S.K. Moon, J.S. Choi, J.G. Kim, D.G. Lee, Biometals 22 (2009) 235–242. [15] P.L. Nadworny, J. Wang, E.E. Tredget, R.E. Burrell, Nanomedicine 4 (2008) 241–251. [16] J.V. Rogers Parkinson, Y.W. Choi, J.L. Speshock, S.M. Hussain, Nanoscale Res. Lett. 3 (2008) 129–133. [17] S. Gurunathan, K.J. Lee, K. Kalishwaralal, S. Sheikpranbabu, R. Vaidyanathan, S.H. Eom, Biomaterials 30 (2009) 6341–6350. [18] K. Kalishwaralal, E. Banumathi, S.R.K. Pandian, V. Deepak, J. Muniyandi, S.H. Eom, Colloid Surf. B: Biointerfaces 73 (2009) 51–57. [19] K. Kalimuthu, R.S. Babu, D. Venkataraman, M. Bilal, S. Gurunathan, Colloid Surf. B: Biointerfaces 65 (2008) 150–153. [20] X. Liu, M. Atwater, Q. Wang, J. Huo, Colloid Surf. B: Biointerfaces 58 (2007) 3–7. [21] A. Nanda, M. Saravanan, Nanomedicine 5 (2009) 452–456. [22] D.J. Freeman, F.R. Falkiner, C.T. Keane, J. Clin. Pathol. 42 (1989) 872–874. [23] G.D. Christensen, W.A. Simpson, J.A. Younger, L.M. Baddour, F.F. Barrett, D.M. Melton, J. Clin. Microbiol. 22 (1985) 996–1006. [24] H.M. Judith, E.K. Daniel, A.O. George, Curr. Protoc. Microbiol. (2005), 1B.1.1–1B.1.17. [25] K.H. Cheng, S.L. Leung, H.W. Hoekman, W.H. Beekhuis, P.G. Mulder, A.J. Geerards, Lancet 354 (1999) 181–185. [26] B.A. Weissman, B.J. Mondino, Contact Lens Anterior Eye 25 (2002) 3–9. [27] S.M.J. Fleiszig, D.J. Evans, Clin. Exp. Optom. 85 (2002) 271–278. [28] A.S. Hoffman, D. Schoen Mack, P. Becker, I. Chattergee, S. Dobinsky, J.K.M. Knobloch, G. Peters, Int. J. Med. Microbiol. 294 (2004) 203–212. [29] N. Nayak, T.C. Nag, G. Satpathy, S.B. Ray, Indian J. Med. Res. 125 (2007) 767–771. [30] A. Mohammed, L. Al-Fattani, J. Med. Microbiol. 55 (2006) 999–1008.