Colloids and Surfaces B: Biointerfaces 79 (2010) 340���344 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f b 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 resis- tant 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 epider- midis 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 pro- longed 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 infec- tions 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 exopolysac- charides 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, Tamil- nadu, India. Tel.: +91 4563 289042 fax: +91 4563 289322. E-mail address: lvsangs@yahoo.com (S. Gurunathan). 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 obste- trician Carl Crede), and for the prevention of infection in burns. When the era of the antibiotics began with the discovery of peni- cillin, 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 impor- tance. 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, sil- ver nanoparticles can be well applied in therapy safely when the effective concentrations of silver nanoparticles on various types of organisms are determined. 0927-7765/$ ��� see front matter �� 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2010.04.014

K. Kalishwaralal et al. / Colloids and Surfaces B: Biointerfaces 79 (2010) 340���344 341 There are many methods available for the synthesis of sil- ver 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 biologi- cally 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 antibi- otic 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 Wild-type of Bacillus licheniformis, P. aeruginosa and S. epider- midis were maintained in nutrient agar as well as sub cultured from time to time in the microbiology laboratory during the study period. 2.2. Synthesis of silver nanoparticles 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 peri- ods. The resulting solution was centrifuged (16,000 rpm, 30 min) and filtered through a 0.22 m filter (Millipore,) to remove cell- debris. 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 6M NA 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). Therefore assuming 100% conversion of all silver ions to silver nanoparticles, N = 3.14 �� 10.5 �� (50.0 �� 10���7)3 �� 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). C = 1 �� 6.023 �� 1023 3837233.003 �� 1 �� 6.023 �� 1023 C = 2.606 �� 10���7 M/L = 260 nM Further, the required concentration are made out from the obtained values. 2.5. Determination of anti-microbial activity by well-diffusion method The AgNPs synthesized from B. licheniformis were tested for anti- microbial 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 for- mation 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 mod- ifications [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