Synthesis Silver Nanoparticles as Antibacterial Against Escherichia coli and Staphlococcus aurous as a Model of Gram- negative and Gram- Positive Bacteria

Bacteria multidrug-resistance (MDR) pathogens led to increasing hospital and community – acquired infections, for which current antibiotic therapies are not effective which represent a growing problem, its well-known that the silver ion and silver nanoparticles have strong effect against microbial. In this study we prepare silver Np in chemical reduction methods and tested it on two type of multidrug resistance bacteria :E-coli and S.aureus. The result showed that the sliver Np has a strong effect against gram-positive and gram-negative bacteria. Key word: Silver Np ,antibacterial , E-coli , S.aureus ةصلاخلا نلالاة قتلالاسريةلا انتنلالا ا ودلالاملا تدنلالا ز لالالا لاد لالار ملالاةوييلا اادنلالااةلا نلالاة تددلالامرة نولالادد قلالاةونلأةلاو قلالاارةةلا ملالا ريريتلا ا تلالاسلا نا ن رودلا سذلا و ا تلاسلا سذلا دلاا ملاةفني قلاةلنمف ااذ اسةل قةلنيلا قةوييلا اادناةلا نة ترفورةلا انجتملا ننف مةمعو انةفشرسةلا لتخ مةيلالا مميلالاشة ملالاثةر رفملالاس ريلالااير رلالايتلا اذلالا لالاف رلالار رلالايي نلالا ريريتلا دلالاا قلالاةلنمف ااذ قلالاةودندلا داولالاةلا ننلالاف مورلالامة ولالاع نلالاةيو وملالاةةندرة ريث لار لامرةة لاودنلادلا رفملاسلا نا لئنلاردلا ايلااوا رلايي اادنلااةمل ملاةونلأةلا نلا ريريتلا نة نيعود مع سرنترخا ررو قةئنةةكلا قلأ رطلنت لاودند قتلنسلاو مجوةلا ن ريريتمل دناة .را ركلا :هيحاتفملا تاممكلا لاودند رفمس مةدولأدملا اا رويةلا ن ريريت و نةي رش ا ن ريريت و لاريريت دناة,


Introduction
In recent years the research has focused on Nobel metal nanoparticles due to unique their chemical and physical properties such as electronic, magnetic, optical properties.Nanoparticles refer to the particles with nano scale size about minimum size 10nm to maximum size 100 nm ,this particle size exhibits large surface area to volume ratio ,the increase in this ratio has changed many properties compared to bulk materials [Marible et al. 2009].
Today Silver nanoparticle one of the most commonly nanoparticles used in medical science , Silver NP play important role in medicine and biological filed ,it has been used widely in many bio applications such as biomedicine ,biosensor, catalysis and used in medicine on burn treatment ,prevention of bacteria colonization on catheters, drug delivery ,etc [Aslan et al. 2005].
The applicability of silver nanoparticles as catalysis depends on the change of their size and stability ,The small surface area to the same ratio of volume means small nano size and that means larger active area of the catalyst .Due to high specific surface area , silver nanoparticles have high strong bacterial activity, against both gram positive and gram negative bacteria.There are many synthetic methods that have been developed to produce silver nanoparticles such as laser ablation, electrochemical method [Rodriguez et al., 2000], chemical reduction methods [Shameli et al.,2011] etc, Methicillin -Resistant Staphylococcus aureus (MRSA) and uropathogenic E. coli (UPEC) strain whichare regarded as the most common pathogen isolated from the patient with UTI.Those two strains are responsible for several difficult -totreat infections in humans also these strains have been also developed through the process of natural selection.[Emody et al.,2003]In this study, we prepare nano silver by chemical reduction methods, and we investigate the influence of silver nano particles against staph and E-coli bacteria

Materials and Methods
Silver nitrate (AgNo 3 ) ,was purchased from Reagent World ,tri sodium citrate,double -distilled deionized water

Silver nano particles preparation Method
Silver Npa was prepared using chemical reduction methods by using tri sodium citrate as a reduction agent ,0.001 M of AgNo 3 was heated to boil ,then 5 ml of tri sodium citrate was added to the solution drop by drop ,the solution were mixed and heated until color changed (pale-yellow) ,then it removed from heat device and it was stirred until cooled to the room temperature.

Bacterial strains
The S.aureus and E. coli bacterial cells were collected and selected from 70 patients (most founding strains were found samples 57 (81.4%)samples with E.coli while S.aurus in the 13(18.6%)samples ) with urinary tract infection who were admittable in Al-Hilla teaching hospital, Babylon during a period from October2015 to January 2016.The two Bacterial isolates were identified as E.coli and S.aureus based on their morphology, Gram-staining.The Vitek 2 system was performed to identify the species level of E. coli isolates.

Antimicrobial Susceptibility Test:
The antimicrobial susceptibility patterns of isolates to different antimicrobial agents was determined and interpreted according to disk diffusion test that was used against 4 antibiotics, The following antimicrobial agents were obtained (from Oxoid, U.K) as standard reference disks as known potency for laboratory use:, trimethoprimsulfamethoxazol (1.25/ 23.75mg), nitrofurantion (300mg), ciprofloxacin (5mg) and Ampicillin (l0mg).

Antibacterial activity
In vitro activity , silver Np against highly multi resistance strains grampositive and gram-negative bacteria was evaluated by inhibition zone testing method by using (MHA) Mueller -Hinton agar [CLSI] for Escherichia-coli and blood agar for Staphyl ococcus aureus ,the inhibition zones were measured in cm after 24 hr of incubation at 35 C 0 .

Result and Discussion
Silver Np was prepared by using chemical reduction method by adding Tri Sodium citrate to aqueous solution of AgNo 3 ,the colorless solution change to pale-yellow color fig ( 1) ,this indicates the formation of nano particles .

Fig (1):color of silver Np solution
The formation and characterization of silver Np was determine by using UV-Vis absorption spectrometer with wave length band about 300-600 nm ,the band of surface plasmon resonance (SPR) determined the morphology of the nanoparticles ,SPR bands are influenced by the morphology, size, and shape of the nanoparticles ,many studies have shown that the spherical silver Np contributes to the absorption band around 400 nm [Stamplecoskis, 2010.],absorption band in this study was around (417) as shown in fig ( 2) wich strongly suggests that silver Np were spherical in the shape.

Fig (2):Absorbance band of silver Np
The size of nanoparticles determines its activity against bacteria because the binding of nanoparticles depends the surface area available for interaction ,when the particle is small this make large surface area available for interaction and this causes more activity of the nanoparticles against bacteria.table (1 The results shows that the nanoparticles have strong activity against gram negative and gram positive bacteria and have more activity against E-coli than Staph.the activity of the Nanoparticles against bacteria have been reported by many studies ,but the mechanism of bactericidal effect of nanoparticles is not very well known, some studies have reported that the electrostatic attraction between positive charge of silver Np and negative charged cell membrane of microorganism [Hamouda, 2000;Dibror et al., 2002;Dragieva et al., 1999],other studies reported that the oxidation react at the surface of nanoparticles silver ion diffusion ,thus causing structural change and finally bacterial will die.(Melinte et al.,2011).
Other reason for bacteria death is that the silver Np may attach to surface membrane and disturb its power function such as permeability and respiration, (Morible et al., 2009) In conclusion silver Np has strong activity against bacteria and this activity is influenced by the size of nanoparticles ,more small particles make large inhibition zones Fig (3): inhibition zones of bacteria