# Introduction iofilms are universal, complex, interdependent communities of surface associated microorganisms. The organisms are enclosed in an exopolysaccharide matrix occurring on any surface, particularly aquatic and industrial water systems as well as medical devices. As such, biofilms are highly relevant for public health (Donlan and Costerton, 2002). Biofilm, likely the predominant mode of device related microbial infection exhibit resistance to antimicrobial agents (Adonizio et al., 2008). They can serve as hides for disease and are often associated with high level antimicrobial resistance of the associated organisms. Biofilms create an environment that enhances antimicrobial resistance. The EPSs of biofilms contain considerable amounts of polysaccharides, proteins, nucleic acids and lipids which are responsible for maintaining structural integrity of the biofilm and provide an ideal matrix for bacterial cell growth. Intermolecular interactions between the functional groups within these macromolecules serve to strengthen the overall mechanical stability of the EPSs and the survivability of the microorganisms..During the past 20 years it has been reported that between 6 and 14% of patients that enter general hospitals develop a nosocomial infection (Vazquez-Argon et al., 2003), i.e., an infection that was not present or incubating at the moment of patient admission at a hospital. Over-all, a large percentage of biofilm-related infections are associated with indwelling medical devices: about 1 million cases-an estimated 60% of nosocomial infections are due to biofilms that have formed on indwelling devices (Darouiche, 2004) Biofilm inhibition carried out in 96 well plates adopting modified method of biofilm spectrophotometric assay (Toole and Kolter, 1998).100?L of cell suspension of the strain thus prepared was added in to 96 well time plate and different concentration of nano particle added and incubated at 37?c for three days after the incubation the liquid culture was removed and 100?L of 1% weight/ volume aques solution of crystal violet was added. Following staining at room temperature for 30 minutes the dye was removed and wells were washed thoroughly, 95% ethanol was added and incubates for 15 minutes the reaction mixture was read spectrophotometrically at 590 nm. Biofilm inhibition (%) was calculated by the following formula % of inhibition = OD in control -OD in treatment # OD in control Catheter was obtained from local medical shop (romo10) the catheter was cut in to 1x1 surface and the cut pieces (5 nos ) were transferred to a beaker containing 20mL of silver nanoparticles suspension with 100?g concentration kept in ultrasonicator for three hours at room temperature, Coating of nanoparticles was confirmed by color change of the catheter surface fine dispersion of particle by scanning electron microscopy and Fontier transform infra red spectroscopy (FTIR) these pieces were used for biofilm inhibition study. # d) Biofilm Inhibition Study The cut pieces was transferred to a test tube containing 5mL of 24 hour culture, the inoculated tubes were kept in C for 3 days (72 hrs) after the incubation period the whole content was aspirated and 5mL of 1% crystal violet was added and incubated at room temperature for 10mins. Crystal violet was removed and successive washing was made using sterile phosphate buffer saline to remove unbound cells or free plantonic cells. After washing, 5mL of ethanol was added kept at room temperature for 15 minutes the reaction mixture was read at 590 nm and the biofilm inhibition was determined as described earlier. III. # Evaluation of Biochemical Composition of Biofilm Matrix # Result and Discussion Biogenesis of silver nanoparticle from leaf extract broth of Azadiracta indica was primarily confirmed by colour change of the reaction mixture from green to brown, plasmon absorption maxima at 420nm by U.V spectrophotometer (Figure 1).Particles morphology was studied by Scanning electron microscopy (SEM).SEM images were recorded by using a Carlzeiss Supra 55 field emission scanning electron microscope equipped with an energy-dispersive spectrum (EDS, oxford instruments) capability. In a SEM setup, the nanoparticulate sample, coated to be conductive (e.g. # Volume XIII Issue III Version I ![importance of yeasts from Candida spp., as common causes of CVC related infections (Ben-Ami et al., 2008). Inhibition of biofilm is considered as drug target and the pharmacological inhibition of biofilm development is now extensively studied for the treatment of various bacterial and fungal infections (Karthick Raja Namasivayam and Allen Roy, 2013). In the present study, anti biofilm effect of biogenic silver nanoparticles coated catheter against clinical isolate of Staphylococcus aureus has been discussed. II. Materials and Methods a) Synthesis of Biogenic Silver Nanoparticles Biogenic silver nanoparticle was synthesized from leaf extract broth of Azadiracta indica (neem) 100gm of dried leaf material was homogenized finely in domestic mixer and 1gm of homogenized material was dissolved in 100ml deionised water and filtered through crude filter paper 50ml of collected filtrate was transferred to 100 ml of beaker and 100ml of 0.1mM silver nitrate was added and the preparation was kept under magnetic stirrer. Conversion of reaction mixture from pale green to dark brown indicates synthesis of silver nanoparticle and further conformation was carried out by uv-visible spectroscopy, Scanning Electron Microscopy and Energy Dispersive Atomic spectroscopy (EDX) the characterized particle was used for further studies. b) Bacterial Stains Clinical isolate of Staphylococcus aureus was obtained from Madurai medical college hospital, Tamil Nadu, India. Bacterial strain was maintained on slope of nutrient agar slant. (Hi media, Mumbai). Nutrient broth was used for inocula preparation. Cultures was inoculated from fresh slopes and incubated with shaking at C for 24 hours. Cells were collected by centrifugation and the collected cell debris washed twice in phosphate buffer saline and suspended to OD520 prior to use in biofilm experiments. c) Biofilm Inhibition Assay](image-2.png "") ![a) Isolation of Biofilm MatrixIsolation of biofilm matrix material from the microtitre plate and catheter was carried out by standard method. Adherent biofilms were transferred to screw cap bottles containing 10 ml distilled water. The bottles were sonicated for 5 min in an ultrasonic water bath and vortexed vigorously for 1 min to disrupt the biofilms. Cell suspensions were then pooled and centrifuged. The collected supernatant used as source for studying biochemical composition mainly protein by Lowry et al and total carbohydrate by Dubois et al.IV.](image-3.png "") 1345![Figure 1 : UV vis spectra of synthesized silver nanoparticles](image-4.png "Figure 1 :Figure 3 :Figure 4 :Figure 5 :") 6![Figure 6 : SEM image of loose cell distrupted Staph.aureus biofilm on the silver nanoparticles coated catheter](image-5.png "Figure 6 :") ![](image-6.png "") ![](image-7.png "") ![](image-8.png "") 1S.NoConcentration ( ?g)Biofilm inhibition (%)11042.122559.435069.047575.55 ___________________________________________________________________________ 100 84.0 2S.NoConcentrationTotalTotal carbohydrateProtein (?g)(?g)11079.070.022545.057.035030.031.947522.014.5510017.58.56 _______________________________________________________________________ Catheter 13.0 5.0 © 2013 Global Journ © 2013 Global Journals Inc. (US) ( )B © 2013 Global Journals Inc. (US) ## Acknowledgement Thanks due to Centre for nanoscience and nanotechnology, Sathyabama University, Chennai, Tamil Nadu, India for SEM, EDAS analysis. gold, palladium), is scanned in a high vacuum chamber with a focused electron beam. The scanning electron microscopy study reveals uniform spherical particles with the size of 50-60nm and the presence of silver in the reaction mixture was further confirmed by EDAS. Biofilm inhibition study clearly revealed all the tested concentration inhibited biofilm of Staphylococcus aureus .Results were represented as inhibition percentage of biofilm development (Table 1). In microtitre plate assay, anti biofilm effect was observed as dose dependent manner. As presented in table 1, silver nanoparticles with 100 µg/ml recorded maximum anti biofilm effect with 84.0 followed by 75.5, 69.0, 59.4 and 42.1 % inhibition at the respective concentration. Coating of biogenic silver nanoparticle was easily identified by color change of catheter (Figure 3) dispersion of nanoparticle on the catheter surface was confirmed by scanning electron microscope which reveals the uniform spherical particles were embedded on the catheter surface with the size of 50 to 60nm (Figure 4). Frontier transform infra red spectroscopy (FTIR) reveals the characteristic changes in the vibrational peaks of coated and non coated catheter (Figure 5). Biofilm inhibition study revealed 87.0 % inhibition during 72 hours of incubation period. Surface topography with SEM reveals complete degeneration of biofilm with weakened cell masses (Figure 6). Similar anti biofilm effect of chemogenic silver nanoparticles coated catheter against clinical isolate of Staphylococcus aureus has been reported (Karthick Raja Namasivayam et al, 2012). Biochemical composition of biofilm matrix total carbohydrate and total protein was also highly reduced. The matrix is one of the most distinctive features of a microbial biofilm. It forms a three dimensional, gel-like, highly hydrated and locally charged environment in which the microorganisms are largely immobilized. Matrix-enclosed micro colonies, sometimes described as stacks or towers, are separated by water channels which provide a mechanism for nutrient circulation within the biofilm the composition of the matrix varies according to the nature of the organism and reduction of the biochemical composition of the biofilm matrix leads to weakening of the biofilm thus facilitate entry of the drugs. In respective concentration of nanoparticles treatment, 70.0, 57.0, 31.9,14.5 and 8.5 ?g of total carbohydrates was recorded under microtitre plate assay Similarly,79.0, 45.0,30.0, 22.0 and 17.5 ?g of protein were recorded Similar reduction of carbohydrate as 5.0 and 13.0 ?g of protein was observed in nanoparticle coated catheter (Table 2). V. * AAdonizio KKong KMathee Antimicrobial Agents and Chemotherapy 52 2008 * RODarouiche N Engl J Med 350. 2004 * MEDavey GO'toole Microbiol Mol Biol Rev 350 2000 * RMDonlan JWCosterton * Clin Microbiol Rev 15. 2002 * Colorimetric method for determination of sugars and related substances MDubois KAGilles JKHamilton PARebers FSmith Anal Chem 28 1956 * Bacterial biofilms: from the natural environment to infectious diseases LHall-Stoodley JWCosterton PStoodley Nature Rev Microbiol 2 2004 * Anti biofilm effect of edicinal plant extracts against clinical isolate of biofilm of Escherichia coli. International journal of pharmacy and pharmaceutical RajaKarthick AllenNamasivayam ERoy Research 5 2 2013 * Biofilm inhibitory effect of silver nanoparticles coated catheter against Staphylococcus aureus and evaluation of its synergistic effect with antibiotics SKarthick Raja Namasivayam MPreethi ArvindBharani RS GeorgeRobin A BavaniLadha International journal of Pharmaceutical and Biological Research 3 2 2012 * Initiation of biofilm formation in pseudomonas fluorescenes WCS365 proceeds via multiple, convergent signaling pathways: a genetic analysis GToole RKolter Mol, Microbiol 28 1998 * PVazquez-Aragon MLizan-Garcia PCascales-Sanchez MTVillar-Canovas DGarcia-Olmo Journal Infect 46 2003 * VonEiff CJansen BKohnen WBecker KManagement Drugs 65 2005