Silver Bullet

Silver Bullet: A New Shine To Antimicrobial Agent, A preface to Silver nanoparticlesAnkita Vishwakarma*, Neelam Balekar, Kuldeep VinchurkarABSTRACT:
This review article generally summarizes the use of silver as an antimicrobial, its mechanism of action, general introduction of silver nanoparticles, synthesis, toxicity, its medical application. Silver is a health additive in traditional Chinese and Ayurvedic medicine. Its action is antibiotic and it is non selective toxic biocide. Silver has been effective against all organism tested and used to treat numerous infection and non infectious condition. Silver is incorporated in wound dressing and used as antibacterial coating in medical devices. Silver ion is bioactive and mainly responsible for bactericidal action. This review also covers the major use of silver in form silver nanoparticle as it has broad spectrum bactericidal and fungicidal activity. Bactericidal nanoparticles are gaining importance because the size of the nanoparticles is similar to biological molecules and are used in in-vitro and in-vivo biomedical studies. Metallic nanoparticles are better nanoparticles with best antibacterial properties as these increase chemical activities due to crystallography surface structure. Nanoparticle of silver brings the significant improvement in the antibacterial activity.

Keywords: Silver, antimicrobial, antibacterial, silver nanoparticles, silver nanoparticle synthesis, toxicity, application
Introduction
Silver is known for its medical properties for over 2000 years1. It has long history in human healthcare and medicine. Silver was used in wound dressing which could be traced back to 18th century during which silver nitrate was used in treatment of ulcers, post surgical infections, in dentistry etc.2 due to the various side effect of silver nitrate colloidal silver was introduced in and was widely used. Colloidal silver usually contains metallic silver particles suspended in solution with less than 10% ionized silver6. Silver was effectively used in wound dressing till 1920, the introduction of antibiotics in 1940 replaced the use of silver and its related compound. The resistant strain of the bacteria helped the emergence of silver. It was used in combination with sulphonamide to create a broad spectrum silver based antibacterial agent2. More recently silver is used in wound dressing with varying level of silver, a variety of silver based dressing have become available eg: Acticoat, Actisorb which offer better therapeutic action. There are advances in creating medical grade silver technology for the synthesis of new, safer and bio available silver compounds. Silver based vascular and urinary catheters are also clinically used. Silver and its compounds are highly toxic to microorganism. Nitrate form of silver is generally used to induce anti microbial effect1, but when silver nanoparticles are used the microbes are exposed to wider surface, silver nanoparticles have use against microbes its action is not fully understood, there are various mechanism through which silver nanoparticles act.

Mechanism of Action
The complete mechanism through which silver acts is still unknown. The few mechanism understood are:
1. Protein inactivation Silver inhibits the bacterial growth by binding with the thiol group in enzymes causing the deactivation of enzymes. Silver forms stable S-Ag bonds with thiol-containing compounds in the cell membrane that are involved in trans-membrane energy generation and ion transport. Silver can take part in catalytic oxidation reactions that result in the formation of disulfide bonds (R-S-S-R). Silver does this by catalyzing the reaction between oxygen molecules in the cell and hydrogen atoms of thiol groups: water is released as a product and two thiol groups become covalently bonded to one another through a disulfide bond. The silver-catalyzed formation of disulfide bonds change the shape of cellular enzymes and affect their function. The silver-catalyzed formation of disulfide bonds leads to changes in protein structure and the inactivation of key enzymes, needed for cellular respiration. It is hypothesized that silver ion bind with 30S ribosomal subunit deactivating the complex and inhibiting the translation of proteins.26
2. DNA association:-Another hypothesis was proposed by Klueh (2000) .The hypothesis proposed that Ag+ enters the cell and get inserted between the purine and pyrimidine base pairs and disrupts the hydrogen bonding between the two anti-parallel strands and denaturing the DNA molecule . Although this has yet to be proved, it has been shown that silver ions do associate with DNA once they enter the cell.26 
3. Action of silver nanoparticles:- The exact mechanism is still unknown but hypothesis suggest that silver nanoparticles have ability to anchor to cell wall of bacteria and penetrate it, causing the structural changes in the cell membrane by forming pits on the cell surface and causing the accumulation of silver nanoparticles.26
Different forms of silver effective at microbial inhibition
1. Silver salts- silver salts such as silver nitrate (AgNO3)
2. Silver zeolites
3. Silver nanoparticlesSilver NanoparticlesNanotechnology is producing nanoproduct and nanoparticles that can have novel and size related physicochemical properties differing from large particle. Nanoparticles size is of similar with the biological molecules and structures can be useful for both invitro and invivo studies.In all nanoparticles metallic nanoparticles are best with antibactericidal properties.Silver is transition metal element with atomic number-47 and atomic mass 107.87.It is a non selective toxic biocide. Silver kills some 650 different disease organisms (Gupta et al).Silver nanoparticles have attracted interest due its unique physical , chemical , biological properties. Silver nanoparticles have distinctive physicochemico properties including high electrical, thermal conductivity, chemical stability. Silver nanoparticles were also found to cause destabilization of outer membrane, collapse of plasma membrane potential and depletion of intracellular ATP. It is an efficient physicochemical system with antimicrobial activities. The silver nanoparticles are generally in the range of 1-100 nm. Silver nanoparticles exhibit broad spectrum bactericidal and fungicidal activity.4
Synthesis of Silver Nanoparticles1. Chemical and Physical synthesis of silver nanoparticles:
The simplest method involves the chemical method of reduction of the metal salt AgBF4 by NaBH4 in water. The nanoparticles which are obtained are of size between 3 to 40nm, which are characterized by transmission electron microscopy (TEM) and UV visible absorption spectroscopy to evaluate the quality.Another method involves sonochemical reduction of an aqueous silver nitrate solution in an atmosphere of argon–hydrogen. The particles recharacterized by TEM, X-ray diffraction, absorption spectroscopy .A microwave synthesis of silver nanoparticles involves the reduction of silver nanoparticles using variable frequency microwave radiation as against conventional method. The method yields higher concentration of silver nanoparticles with the same temperature and exposure.1
2. Biological synthesis of silver nanoparticle:
Chemical and physical method of synthesis silver nanoparticle is expensive and involve the use of toxic and hazardous chemicals. The need for the environmental and economically safe way to synthesize these nanoparticles led to the search of new method which involves the biomimetic production of silver by biological method. The three major sources of synthesizing silver nanoparticles involves bacteria, fungi and plant extract.1
A. Silver synthesizing bacteria:
The nitrate reductase enzyme converts nitrate into nitrite. In invitro synthesis of silver using bacteria the presence of alpha nicotinamide adenine dinucleotide phosphate reduced form (NADPH) – dependent nitrate reductase would remove the downstream process step. During reduction nitrate is converted into nitrite and electron is transferred to the silver ion. This is observed in Bacillus licheniformis which secrete NADPH and NADPH dependent enzymes like nitrate reductase that converts Ag+ to Ag0.1
S.No. Bacteria Particle size (nm)
1. P. stutzen200
2. Bacillus megaterium46.9
3. Plectonema borynum1-200
4. Enterobacter cloacae 50-100
5. Escherichia coli 5-25
Table 1 . Silver synthesizing bacteria and synthesized particle size 1
B. Silver synthesizing fungi:
Fungi can large amounts of nanoparticles because they can secrete large amount of proteins which directly translate to higher productivity of nanoparticles. It involves the trapping of Ag+ ions at the surface of fungal cell and subsequent reduction of the silver ions by the enzyme present in the fungal system .The enzymes like napthaquinones and anthroquinones facilitates the reduction. Exact mechanism involved is not known but the above mentioned phenomenon is responsible for the process but the process is very slow.1
S.No. Fungi Particle size (nm)
1. Vertcillium sp. 25
2. F.oxyporum20-50
3. Aspergillus fumigates 5-25
4. Aspergillus flavus7-10
5. Fusarium solani5-35
Table 2 . Silver synthesizing fungi and synthesized particle size 1
C. Silver synthesizing plants:
The advantage using plant extracts for silver nanoparticles synthesis is that they are easily available and non toxic in nature. The main is plant assisted reduction by phytochemicals like terpenoids, flavones, ketones, amides etc. Previous studies explain that Xerophytes contain emodins an anthraquinone that undergo tautomerization leading to the formation of silver nanoparticles .Phytochemicals are involved directly in reduction of ions and formation of silver nanoparticles.1
S.No. Plant Particle size (nm)
1. Medicago sativa 2-20
2. Azadirachta indica50
3. Aloe vera15-20
4. Cinnamomum camphora leaf 55-80
5. Cinnamomum zeylanicum bark 50-100
Table 3. Silver synthesizing plant and synthesized particle size 1
Biological properties of silver nanoparticles:
Silver nanoparticles are used in medicine due to its antibacterial, antifungal, antiviral, anti-inflammatory, osteoinductive effect and property of wound healing.

1. Antibacterial properties of silver nanoparticles:
Antibiotics bind to specific chemical targets of bacteria, this binding specifically narrows the number of bacterial species that are vulnerable to a specific antibiotic later on multiple dosing bacteria grows resistant towards the antibiotics. Silver is used as an alternative because it is an antiseptic that targets a broad spectrum of gram positive and gram negative and vancomycin resistant strains. Due to greater surface to mass ratio silver nanoparticles offer greater active surface, higher solubility and chemical reactivity. Antibacterial activity of silver nanoparticles was dependent on the surface area with nanoscales showing lager surface area. Silver nanoparticles show antibacterial activity towards S.aureus, S.epidermidis, Enterococcus faecalis, Enterococcus faecium, E.coli, P.aeruginosa and K.pneumonia.3
2. Antifungal properties of silver nanoparticles:
The repetitive long term administration of antifungal drugs leads to the fungal resistance, generally by Candida species. Silver nanoparticles exhibits antifungal activity towards C. tropicalis, C.krusei, C.glabrata, C.albicans. Silver nanoparticles synthesized from aqueous raspberry extract acted as growth inhibitor against Cladosporium cladosporioides and Aspergillus niger. Higher concentration of silver nanoparticles induces reduction in fungal rate.3
3. Antiviral properties of silver nanoparticles:
Silver nanoparticles act as broad spectrum agent against a variety of viral strains. The antiviral activity of silver nanoparticles has been studied against HSV-1, HSV-2, Hepatitis. The mode of action of silver nanoparticles occurs during the early phases of viral replication.3
4. Anti-inflammatory properties of silver nanoparticles:
Silver nanoparticles coated wound dressing decreases the level of proinflammatory cytokines transforming growth factors-(TGF) beta and tumor necrosis factor (TNF) alpha.3
5. Osteoconductivity and Osteoinductivity of silver nanoparticles based materials:
AgNP(silver nanoparticles) implant induces osteogenesis while suppressing the bacterial survival in contaminated rat femoral canals with no cytotoxicity. The composite grafts of AgNP/PLGA display the osteoconductive properties as they did not inhibit adherence, proliferation. These findings require more thorough research but they promise therapeutic materials for orthopaedic surgery.3
Literature surveys and their interpretation regarding silver nanoparticles:
DVM et al. studied the low concentration of silver nanoparticles and it was found to exhibit microbicidal effect on yeast and E.coli. However on Staphyllococcus aureus (gram positive bacteria) mild antimicrobial effect was seen. 4,7
Ping Li and Juan Li studied the bactericidal action of silver nanoparticles with amoxicillin on E.coli was studied. And compared it to the individual treatment, when amoxicillin and silver nanoparticles were combined, prominent bactericidal activity of silver nanoparticles was observed. 4,8
SK Gogoi et al. performed examination which involves the synthesis of silver nanoparticles by sodium borohydride method and was evaluated on recombinant E.coli bacteria. The study concludes that silver nanoparticles above a certain concentration were not only bactericidal but also reduces the sizes of the treated bacteria compared to untreated ones. No direct effect on DNA/protein was observed. 4,9
Sharma et al observed the antibacterial activity of silver nanoparticles and its modified form by polymers and surfactant against gram positive and gram negative bacteria. 4,10
Anh-Tuan Le et al. experimented on oleic acid stabilized silver nanoparticles which were obtained by the green chemical synthetic method, appeared to possess high antibacterial activity against S.aureus. 4,11
Wang et al. performed experiment in which silver ions were exchanged with titanium phosphate film by ion exchange process, which was effective in prohibiting growth of E.coli and could be used as coatings. 4,12
M Liong et al. the silver nanocrystals encapsulated in mesoporous silica nanoparticles which possess antibacterial activity against gram positive and gram negative bacteria. 4,13
Panacek et al. studied antifungal activity of silver nanoparticles for pathogen Candida sp. which were prepared by Tollens process. And by the determination of MIC (minimum inhibitory concentration), MFC (Minimum fungal concentration) and time dependency of yeast growth inhibition, it was observed that at concentration as 0.21 mg/l of silver, silver nanoparticles exhibited inhibitory effects of silver nanoparticles against tested pathogen. 4,14
Leung et al. studied antimicrobial activity of silver nanoparticles consisting of titanium dioxide and silver nitrate on gram negative and gram positive bacteria and it was observed that 1/128 and 1/512 were the MIC of nanoparticles against E.coli and S.aureus respectively. 4,15
By Kirby-Bauer method, silver nanoparticles was synthesized by chemical reduction from aqueous solution of silver nitrate containing a mixture of hydrazine hydrate and sodium citrate as reductants and sodium dodecyl sulphate as stabilizer was found to possess antibacterial activity against E.coli, S.aureus, Pseudomonas aeruginosa. 4,16
Muthuswamy and Krishnamurthy synthesized silver nanoparticles from the water soluble organics of Curcuma longa tuber and extract, and the MBC (minimum bactericidal concentration) for E.coli BL-21 strain was 50mg/l. 4,17
Musarrat et al. isolated a fungal strain KSU-09 from the roots of date palm (Phoenix dactylifera) which was identified as Amylomyces rouxii and it was found to synthesize silver nanoparticles, have antimicrobial activity against Shigella dysentriae type 1, S.aureus, Citrobacter sp., E.coli, P.aeruginosa, B.subtilis, C.albicans, Fusarium oxyporum. 4,18
Similarly, silver nanoparticles synthesized from Mentha piperita leaves extract was found to possess antimicrobial activity against clinically isolated human pathogens. (Mubarakali et al) 4, 19
Again, biologically synthesized silver nanoparticles from Acalypha indica leaf extract has antimicrobial activity against Vibrio.cholera, E.coli. MIC was 10ug/ml for both pathogens (Krishnaraj et al). 4,20
Kalimuthu et al. studied and observed that biologically synthesized silver nanoparticles exhibited a potential antibiofilm formation activity that was tested in vitro on biofilms formed by P.aeruginosa and S.epidermidis during 24 hour treatment, treating these organisms with silver nanoparticles resulted in more than 95% inhibition of biofilm formation. 4,21
Shivaji et al. studied antibacterial activity of silver nanoparticles synthesized from psychrophillic bacteria which were analyzed against Arthrobacter kergulesis , A.gangotriensis and B.indicus. And it was found that the lowest concentration is 2ug/ml at which they arrest the bacterial growth as indicted by agar diffusion method. 4,22
Mirzajani et al. studied the antibacterial activity of silver nanoparticles on S.aureus PTCC1431 and suggested that concentration of silver nanoparticles above 8ug/ml results in the release of muramic acid into the medium that cause cell wall destruction. 4,23
Sondi et al. studied that E.coli cells treated with silver nanoparticles found to be accumulated in bacterial membrane which results in the increase of permeability and later in cell death. 4, 24, 25
DVM et al. used electron resonance spectroscopy to investigate the effect of silver nanoparticles to microbes and found that they exert their effect by generation of free radicals. 4,7
Thombre et al. studied silver nanoparticles synthesized from Cinnamomum tamala or Indian bay leaf exhibit antibactericidal action against extremely resistant haloarchaea and MIC for it was found to be between 300-400 ug/ml. 5
Use of silver
1. Wound dressing: Medical grade silver containing dressing approved by FDA/EPA includes Silvadene (Marion Laboratories), Curad silver (Beiersdorf AG), Actisorb 220(Johnson & Johnson)6.

2. Endotracheal tubes: Endotracheal tubes are used by patients needing ventilator-assisted breathing. Silver coatings on the inside of endotracheal tubes delay the appearance of bacteria on the insides of these tubes.26
3. Surgical mask: Surgical masks coated with silver nanoparticles were capable of a 100% reduction in viable E. coli and S. aureus cells after incubation.26
4. Silver nanoparticles are used in bone cements that are used in bone replacement therapy 1.

5. Nanosilver are also used in biosensing for biosensing large number of protein 1.

6. Nanosilver also used in bioimaging using its plasmonic properties 1.

7. Axenohl (Axen30) is silver dihydrogen citrate disinfectant which kills sensitive strain bacteria in 30 seconds and resistant strains, such as MRSA, in 2 minutes 6.

Toxicity of silver
The major side effect known of silver include the condition of Argyria which is identified by the development of bluish grey skin known to occur at the lengthy exposure to silver6. Also silver wound dressing which release low level of silver ions are more dangerous in terms of resistance if silver ion concentration is sublethal. Faster acting dressing present less risk because organism is most likely to be killed therefore eliminating the enrichment of the resistant population through growth and division. Also nanosilver aggregates are more cytotoxic than asbestos1. Nanosilver can induce the proliferation of and cytokine expression by peripheral blood mononuclear cells. Nanosilver also shows severe toxic effects on the male reproductive system1.

Conclusion
The resistance towards the antibiotics is a major issue and alarming concern. There is need for a cheap broad active agent that can be used against micro-organisms. Silver is an excellent antimicrobial agent and has been used for a long time, but there is need for silver MIC (minimum inhibitory concentration) level and breakpoints to be developed, studied and standardized. The antimicrobial properties of silver are due to its ionized form, Ag+, and its ability to cause damage to cells by interacting with thiol-containing proteins and DNA. Silver nanoparticles are a form of silver of particular interest because of their easy production, high antimicrobial activity, and ability to be incorporated into a diverse range of products. With the ever increasing number of antibiotic-resistant strains of bacteria and silver’s low toxicity to humans, the use of silver as an antimicrobial agent are relevant to many fields of study and industry. Recently it has been concluded that silver nanoparticles acts bactericidal, virucidal, and promotes wound healing. But nonetheless conclusive safety is not studied widely, therefore additional testing of silver nanoparticles is need to be studied before they can be used in clinical applications. Also, studies prove that silver nanoparticles can induce ecological problems and cause disturbance in ecosystem, so there is need to synthesize the silver nanoparticle in an effective and efficient way.

REFERENCES:
S. Prabhu, E. K. Poulose. Silver nanoparticles: mechanism of antimicrobial action, synthesis, medical applications, and toxicity effects. International nanoletters 2012, 2:32.

I. Chopra. The increasing use of silver based products as antimicrobial agent: a useful development or cause of concern? Journal of Antimicrobial Chemotherapy, 2007. 59: 587-589.

M. Murphy, K. Ting, X. Zhang, C. Soo, Z. Zheng. Current development of silver nanoparticle preparation, investigation, and application in the field of medicine. Journal of Nanomaterial, 2015, Article ID 696918, 12 pages.
A. Vedpriya, K. Ratika, M. Kaur, A. Goyal. Silver Nanoparticles as a potent Antimicrobial Agent: A review, Journal of Nanomaterials 2011, 3: 118-124.

Thombre SR, Shinde V, Thaiparamil E, Zende S, Mehta S. Antimicrobial activity and mechanism of inhibition of silver nanoparticles against extreme Halophilic archaea. Journal of Frontiers in Microbiology, 2016, 7:1424.

A. Melaiye, W. J.Youngs. Silver and its application as an antimicrobial agent, Expert opinion on Therapeutic Patents,2005.15:2, 125-130
J. DVM, E. Kuk, K. N. Yu, S. H. Kim, S. J. Park, H. J. Lee, J. Kim, Y. H. Park, C. Hwang, Y. Kim, Y.Lee, D. Jeong and M. Cho. 2007. Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine. 3(1): 95-101.

P. Li, J. Li, C. Wu, Q. Wu and J. Li. Synergistic antibacterial effects of beta lactam antibiotic combined with silver nanaoparticles. Nanotechnology. 2015, 16(9): 12.

S. K. Gogoi, P. Gopinath, A. Paul, A. Ramesh, S. S. Ghosh and A. Chattopadhyay. Green fluorescent protein-expressing E. coli as model system for investigating antimicrobial activities of silver nanoparticles. Langmuir. 2006, 22(22): 9322-9328.

V. K. Sharma, R. A.Yngard and Y. T. Lin. Silver nanoparticles: green synthesis andtheir antimicrobial activities. Advances in Colloid and Interface Science.2009, 145(1-2): 83-96.

A.T. Le,L.T. Tam, P. D. Tam, P. T Huy, T. Q. Huy , N. V. Alieu , A. A. Kudrinskiy and Y. A. Krutyakov. 2010. Materials Science and Engineering: 30(6): 910-916.

Q. Wang, H. Yu, L. Zhong, J. Liu, J. Sun and J. Shen. Incorporation of silver ions in to ultrathin titanium phosphate films: in situ reduction to prepare silver ions and their antibacterial activity. Cem-Mater.2006. 18(7): 1988-1994.

M. Liong, B. France, K. A. Bradley and J.I. Zink . Antimicrobial activity of silver nanocrystals encapsulated in mesoporous silica nanoparticles. Advance of Material.2009. 21(17): 1684-1689.

A. Panacek, M. Kolar, R.Vercerova, Prucek R, Soukupova J, Krytof V, Hamal P, Zboril R and Kvitek L. 2009. Antifungal activity of silver nanoparticles against Candida sp. Biomaterial. 30: 6333-6340.

Y. Li, P. Leung, L. Yao, Q. W. Song and E. Newton. Antimicrobial effect of surgical masks coated with nanoparticles. J Hospital Infection.2006. 62(1): 58-63.

M. Guzman, J. Dille and S. Godet. Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram negative bacteria.Nanomedicine: Nanotechnology, Biology and Medicine. 2011.

S. Muthuswamy, S. Krishnamurthyand Y.Yeoung-Sang. Immobilization of silver nanosynthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity. Bioresource technology.2001. 101: 7958-7965.

J. Musarrat, S.Dwivedi, B. R.Sinh, A. A. Al-Khedhairy, A.Azam, A. Naqvi. Production of antimicrobial silver nanoparticles in water extracts of fungus Amylomycesrouxii strain KSU-09. Bioresource Technology.2010. 101: 8772-8776.

D. Mubarakali, N. Thajuddin, K. Jegananthan and M. Gunasekaran. Plant extracts mediated synthesis of silver and gold nanoparticles and its antibacterial activity against clinically isolated pathogens. Colloids and Surfaces B: Biointerfaces. 2011. 35: 360-365.

C. Krishnaraj, E. G. Jagan, S. Rajasekar, Selvalumar P, Kalaichelvan PT and Mohan N. Synthesis of silver nanoparticles using Acalyphaindica leaf extracts and its antibacterial activity against water borne pathogens. Colloids and Surfaces B: Biointerfaces.2009 76: 50-56.

K. Kalimuthu, B. Selvaraj, Pandian SRK, Venkataraman D and Sangiliyandi G.Silver nanoparticles impede the biofilmformation by P. aeruginosa and S. epidermidis. Colloids and Surfaces B: Biointerfaces. 2010.79: 340-344.

S. Shivaji, S. Madhu and S. Singh. Extracellular synthesis of antibacterial silver nanoparticles using psychrophillic bacteria. Process Biochemistry. 2011. 46: 1800-1807.

F. Mirzajani, A. Ghassempour, A. Aliahmadi and M. Esmaeili. Antibacterial effect of silver nanoparticles on Staphylococcus aureus. Research in Microbiology. 2011 162: 542-549.

I Sondi and B. S. Sondi. Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for gram negative bacteria. J. Colloid and Interface Science.2004. 275(1): 177-182.

J. B. Wright, K. Lam, D. Hanson, R.E. Burrell. Am J Inf Cont. NA 1995.7:627.

https://microbewiki.kenyon.edu/index.php/Silver_as_an_Antimicrobial_Agent