Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Mndez-Vilas (Ed.

Nanoparticles and their potential application as antimicrobials

Ravishankar Rai V *and Jamuna Bai A
Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore, India. * Email: raivittal@gmail.com Emerging infectious diseases and the increase in incidence of drug resistance among pathogenic bacteria have made the search for new antimicrobials inevitable. In the current situation, one of the most promising and novel therapeutic agents are the nanoparticles. The unique physiochemical properties of the nanoparticles combined with the growth inhibitory capacity against microbes has led to the upsurge in the research on nanoparticles and their potential application as antimicrobials. From centuries metals such as silver have been used for treating burns and chronic wounds, and copper has been used to make water potable. It is quite evident that some of the metallic compounds possess antimicrobial property. Recently, the confluence of nanotechnology and biology has brought to fore metals in the form of nanoparticles as potential antimicrobial agents. Nanoparticles have unique and well defined physical and chemical properties which can be manipulated suitably for desired applications. Moreover, their potent antimicrobial efficacy due to the large surface area to volume ratio has provided them an edge over their chemical counterparts which are facing the problems of drug resistance. This review focuses on the properties of different types of metallic nanoparticles such as copper, aluminium, gold, silver, magnesium, zinc and titanium nanoparticles. The mechanism of action of nanoparticles as bactericidal, antifungal and antiviral agents will be highlighted in this study. The potential application of nanoparticles will be also reviewed. The application of nanoparticles as antimicrobials is gaining relevance in prophylaxis and therapeutics, in medical devices, food industry and textile fabrics. The problems related to toxicity of nanoparticles will be addressed in brief. Keywords: Nanoparticles, antimicrobials agents, therapeutics, drug resistance.

1. Introduction
The emerging infectious diseases and the development of drug resistance in the pathogenic bacteria and fungi at an alarming rate is a matter of serious concern. Despite the increased knowledge of microbial pathogenesis and application of modern therapeutics, the morbidity and mortality associated with the microbial infections still remains high [1]. Therefore, there is a pressing demand to discover novel strategies and identify new antimicrobial agents from natural and inorganic substances to develop the next generation of drugs or agents to control microbial infections. Prior to the extensive use of chemotherapeutics in modern health care system, inorganic antimicrobials such as silver and copper were used since ancient times to treat microbial infections [2]. In the recent times, the advances in the field of nanosciences and nanotechnology has brought to fore the nanosized inorganic and organic particles which are finding increasing applications as amendments in industrial, medicine and therapeutics, synthetic textiles and food packaging products [3]. Nanoparticles usually ranging in dimension from 1-100 nanometers (nm) have properties unique from their bulk equivalent. With the decrease in the dimensions of the materials to the atomic level, their properties change. The nanoparticles possess unique physico-chemical, optical and biological properties which can be manipulated suitably for desired applications [4]. Moreover, as the biological processes also occur at the nanoscale and due to their amenability to biological functionalization, the nanoparticles are finding important applications in the field of medicine [5]. The nanoparticles are broadly grouped into organic and inorganic nanoparticles. The latter have gained significant importance due to their ability to withstand adverse processing conditions [6]. Currently, the metallic nanoparticles are thoroughly being explored and extensively investigated as potential antimicrobials. The antimicrobial activity of the nanoparticles is known to be a function of the surface area in contact with the microorganisms. The small size and the high surface to volume ratio i.e., large surface area of the nanoparticles enhances their interaction with the microbes to carry out a broad range of probable antimicrobial activities. Metal nanoparticles with antimicrobial activity when embedded and coated on to surfaces can find immense applications in water treatment, synthetic textiles, biomedical and surgical devices, food processing and packaging [7]. Moreover, the composites prepared using metal nanoparticles and polymers can find better utilization due to the enhanced antimicrobial activity. In this review, we focus on the role of various metallic nanoparticles as potential antimicrobials and the possible mechanism of their inhibitory actions. The increasing application of nanoparticles as antimicrobials in industries, medicine, cosmetics, textiles and food packaging which requires the assessment of the toxicity and risks associated with these particles will also be reviewed.

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2. Properties of Nanoparticles
The dimension of matter important in nanoscience and nanotechnology is typically on the 0.2- to 100-nm scale (nanoscale). The properties of materials change as their size approaches the nanoscale. Further, the percentage of atoms at the surface of a material becomes more significant [8]. Bulk materials possess relatively constant physical properties regardless of their size, but at the nanoscale this is often not the case. As the material becomes smaller the percentage of atoms at the surface increases relative to the total number of atoms of the material bulk. This can lead to unexpected properties of nanoparticles which are partly due to the surface of the material dominating over the bulk properties. At this scale, the surface-to-volume ratios of materials become large and their electronic energy states become discrete, leading to unique electronic, optical, magnetic, and mechanical properties of the nanomaterials. In general, as the size of inorganic and organic materials decreases towards the nanoscale, their optical and electronic properties largely varies from the bulk material at the atomic/molecular levels and is size and shape dependent. The various size dependent properties that can be observed are quantum confinement in semi-conductor particles, surface plasmon resonance in noble metal particles and super paramagnetism in magnetic materials. Thus, the crystallographic surface structure and the large surface to volume ratio make the nanoparticles exhibit remarkable properties. Moreover, the increased catalytic activity due to morphologies with highly active facets and the tailoring of its synthesis as per the requirement makes the nanoparticles an attractive tool to solve various technological problems [9-10]. In the field of medicine, nanoparticles are being explored extensively because of their size dependant chemical and physical properties. The size of nanoparticles is similar to that of most biological molecules and structures. This makes them an interesting candidate for application in both in vivo and in vitro biomedical research. The result of their integration in the field of medicine has led to their application mainly in targeted drug delivery, imaging, sensing, and artificial implants. Another interesting avenue for their exploration in medicine is their use as antimicrobials to target highly pathogenic and drug resistant microbes. But, for the application of nanoparticles in biology, biocompatibility is a highly desired trait. Biocompatibility is the materials ability to perform medically without exertion of undesired local or systemic effects [11].

3. Nanoparticles as antimicrobials
3.1 Antibacterial activity of nanoparticles 3.1.1 Silver nanoparticles Silver compounds have been used to treat burns, wounds and infections [12]. Various salts of silver and their derivatives are used as antimicrobial agents [13-14]. Recent studies have reported that nanosized silver particles exhibit antimicrobial properties [15-16]. Nanoparticles of silver have been studied as a medium for antibiotic delivery, and to synthesize composites for use as disinfecting filters and coating materials [17-19]. Several mechanisms have been proposed to explain the inhibitory effect of silver nanoparticles on bacteria. It is assumed that the high affinity of silver towards sulfur and phosphorus is the key element of the antimicrobial effect. Due to the abundance of sulfur-containing proteins on the bacterial cell membrane, silver nanoparticles can react with sulfur-containing amino acids inside or outside the cell membrane, which in turn affects bacterial cell viability. It was also suggested that silver ions (particularly Ag+) released from silver nanoparticles can interact with phosphorus moieties in DNA, resulting in inactivation of DNA replication, or can react with sulfur-containing proteins, leading to the inhibition of enzyme functions [20-21]. The general understanding is that Ag nanoparticle of typically less than 20 nm diameters get attached to sulfur-containing proteins of bacterial cell membranes leading to greater permeability of the membrane, which causes the death of the bacteria [22]. The dose dependent effect of silver nanoparticles (in the size range of 10-15 nm) on the Gram-negative and Grampositive microorganisms has been studied [23]. At micromolar levels of Ag+ ions have been reported to uncouple respiratory electron transport from oxidative phosphorylation, inhibit respiratory chain enzymes, or interfere with the membrane permeability to protons and phosphate [24-26]. In addition, higher concentrations of Ag+ ions have been shown to interact with cytoplasmic components and nucleic acids [27-28]. The effect of silver nanoparticles on the cell morphology of Escherichia coli and Staphylococcus aureus has been studied using TEM, SEM and X-ray microanalyses [29-30]. It was revealed that treatment with the silver ions results in similar morphological changes in both the Gram positive and Gram negative bacteria. The cytoplasmic membrane detaches from cell walls and an electron-light region containing condensed deoxyribonucleic acid molecules appears in the centre of the cell. The inhibitory activity of silver ions is higher in case of Gram negative bacteria. This might be due to the thickness of the peptidoglycan layer in Gram-positive bacteria cell wall which may prevent to some extent, the action of the silver ions [29]. The formation of electron-dense granules containing silver ions and sulphur ions in the cytoplasm of the bacterial cell suggests that the possible mechanism of action of silver nanoparticles may be due to the

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interaction of silver ions with nucleic acids and impairment of DNA replication which results in loss of cell viability and eventually resulting in cell death [31]. The bactericidal effect of silver nanoparticles typically ranging from 2 to 5 nm has been investigated using green fluorescent protein (GFP)-expressing recombinant Escherichia coli [32]. Apart from the conventional viability tests, the morphological changes of the fluorescent bacteria and electrophoretic analysis of cellular DNA and protein migration profiles were performed to establish the effect of silver nanoparticles on GFP bacteria. The silver nanoparticles of less than 10 nm diameters attached to the bacterial cell wall causes perforation of the cell wall, which leads to the cell death. This study suggests that the mode of action of silver nanoparticles is that the NPs get attached to the sulfur-containing proteins on the bacterial cell wall, leading to increased permeability of the membrane, finally causing cell death [32]. There are also studies reporting that metal ions induce generation of intracellular reactive oxygen species in bacterial cells [33]. Ag ions released by active surfaces of silver nanoparticles and silver oxide present on the surfaces of these nanoparticles are reported to be the actual biocidal agents [34]. The silver ions enter the bacterial cells, where they are reduced as the cell attempts to remove them from the cell interior, eventually leading to cell destruction [22]. It has been recently demonstrated that silver nanoparticles of less than 10 nm diameter make pores on the bacterial cell walls. The cytoplasmic content is released to the medium, which leads to cell death without affecting the intracellular and extracellular proteins and nucleic acids of the bacterium [34]. It has been demonstrated that composites of silver nanoparticles with polymer results in the improvement of antimicrobial activities of silver nanoparticles at lower concentrations [35-37]. Among the polymers chitosan, a cationic polysaccharide composing randomly distributed (1,4)-linked 2-amino-2-deoxy--D-glucose units has been reported to be used as such or in the form of composite with silver nanoparticles with high antimicrobial efficacies. It is generally accepted that polycationic chitosan can bind with negatively charged cell membranes, which will then lead to decrease in the osmotic stability of the cell, followed by subsequent leakage of intracellular constituents. The chitosan silver nanoparticles have enhanced antimicrobial activity than its individual components i.e. chitosan and silver. In the composite, the essential function of the positively charged chitosan matrix was to capture negatively charged bacteria on its surface, while small sized Ag NPs created pores on bacterial wall, thereby causing rapid disintegration of the bacteria [38]. A proteomic approach (two dimensional electrophoresis and proteins identification by mass spectrometry) was used to investigate the mode of antibacterial action of nano-Ag against E. coli [39]. The proteomic studies revealed for the first time, several primary actions of nano-Ag in E. coli cells, namely in envelope protein processing, outer membrane permeability, plasma membrane potential and energization. The proteomic analyses revealed that just a short exposure of E. coli cells to nano-Ag resulted in alterations in the expressions of a number of envelope proteins and heat shock proteins which are usually induced in a variety of stress conditions. The envelope proteins (OmpA, OmpC, OmpF, OppA, and MetQ) are integral outer membrane or periplasmic components guarding against the entry of foreign substances. In particular, the expression of the heat shock proteins IbpA and IbpB is stimulated during the over expression of heterologous proteins that are associated inclusion bodies [40-43]. The treatment with nano-Ag destabilizes the outer membrane and disrupts the outer membrane barrier components such as lipopolysaccharide or porins, culminating in the perturbation of the cytoplasmic membrane. The proteomic signatures of nano-Ag treated E. coli cells are characterized by an accumulation of envelope protein precursors. This indicates that nano-Ag may target the bacterial membrane, leading to a dissipation of the proton motive force. Although the detailed mechanism by which nanoparticles with a diameter of 10 nm can penetrate and disrupt the membranes remains to be elucidated, electron microscopy and optical imaging results suggest that nano-Ag penetrate the outer and inner membranes of the Gram negative bacteria, with some nanoparticles found intracellularly [41]. Nano-Ag and Ag+ ions share a similar membranetargeting mechanism of action. But the effective concentrations of nano-Ag and Ag+ ions are at nanomolar and micromolar levels, respectively. Nano-Ag appears to be significantly more efficient than Ag+ ions in mediating their antimicrobial activities [44]. Size-controlled silver colloid nanoparticles generated using a one-step modified Tollens process was assessed for antimicrobial activity against drug resistant pathogens [45]. The pH and type of reducing saccharide of the reaction system were found to influence the size of particles. A wide range of particle size, particularly 25-100 nm with narrow size distributions were obtained using four different saccharides. Nanoparticles with size less than 25 nm have exhibited minimum inhibitory concentration of 6.75-54 g/mL whereas 25 nm size particles showed MIC in the range of 1.6913.5 g/mL against including highly multiresistant bacteria such as methicillin-resistant Staphylococcus aureus, methicillin-resistant coagulase-negative staphylococci (e.g., Staphylococcus epidermidis), vancomycin-resistant Enterococcus faecium, and ESBL-positive Klebsiella pneumoniae. This is an important result, particularly when antibiotic resistance among bacterial species is increasing at an alarming rate and very few alternative options are available to address the issue. Silver nanoparticles have been evaluated for their antimicrobial activities against a wige range of pathogenic organisms [46-51]. The highest sensitivity was observed against Methicillin resistant Staphylococcus aureus (MRSA) followed by Methicillin resistant Staphylococcus epidermidis (MRSE) and Streptococcus pyogenes. A moderate antimicrobial activity was observed in case of the gram negative pathogens Salmonella typhi and Klebsiella pneumonia [52].

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The size of the particle plays a central role in antimicrobial activity [22] The colloidal silver particles, with variable + sizes (44, 50, 35, and 25 nm), synthesized by the reduction of [Ag (NH3)2] complexes with carbohydrates were tested for antimicrobial activity [45]. The antibacterial activity was particle size dependent. The silver nanoparticles also exhibit a shape-dependent interaction with the bacterial cells. The truncated triangular silver nanoplates displayed the strongest biocidal action against E. coli, when than the spherical and rod-shaped nanoparticles [53]. Small particles exhibited higher antimicrobial activity than big particles. This result can be due to high particle penetration when these particles have smaller sizes. The antibacterial properties are related to the total surface area of the nanoparticles. Smaller particles with larger surface to volume ratios have greater antibacterial activity [ 54-56]. 3.1.2 Gold nanoparticles The therapeutic use of gold can be traced back to the Chinese medical history in 2500 BC. Red colloidal gold is still used in the Indian Ayurvedic medicine for rejuvenation and revitalization during old age under the name of Swarna Bhasma (Swarna meaning gold, Bhasma meaning ash) [57-58]. Gold also has a long history of use in the western world as nervine, a substance that could revitalize people suffering from nervous conditions. In the 16th century gold was recommended for the treatment of epilepsy. In the beginning of the 19th century gold was used in the treatment of syphilis. Following the discovery of the bacteriostatic effect of gold cyanide towards the tubercle bacillus by Robert Koch, gold based therapy for tuberculosis was introduced in 1920s [59]. The major clinical uses of gold compounds are in the treatment of rheumatic diseases including psoriasis, juvenile arthritis, planindromic rheutamitism and discoid lupus erythematosus [60]. Au particles are particularly and extensively exploited in organisms because of their biocompatibility [61-62]. Gold nanoparticles (Au) generally are considered to be biologically inert but can be engineered to possess chemical or photothermal functionality. On near infrared (NIR) irradiation the Au-based nanomaterials, Au nanospheres, Au nanocages, and Au nanorods with characteristic NIR absorption can destroy cancer cells and bacteria via photothermal heating. Au-based nanoparticles can be combined with photosensitizers for photodynamic antimicrobial chemotherapy. Au nanorods conjugated with photosensitizers can kill MRSA by photodynamic antimicrobial chemotherapy and NIR photothermal radiation [63-64]. A hydrophilic photosensitizer, toluidine blue O was conjugated on the surface of Au nanorods for photodynamic antimicrobial chemotherapy. The Au nanorods served as both photodynamic and photothermal agents and inactivated MRSA. The combined effect of PACT and hyperthermia has enhanced antimicrobial effect of gold nanoparticle. The study clearly showed that gold nanorods conjugated with a hydrophilic photosensitizer such as toluidine blue O act as dual-function agents in photodynamic inactivation and hyperthermia against methicillin-resistant Staphylococcus aureus [65-66]. Light absorbing gold nanoparticles conjugated with specific antibodies have also been exploited to photothermally kill Staphylococcus aureus by using laser [67]. Recent studies have focussed on functionalising the gold nanoparticles as phothermal agents for hyperthermically killing pathogens [68-70] The efficacy of the antibacterial activity of gold nanoparticles can be increased by adding antibiotics [71].The antimicrobial activity of the antibiotic vancomycin was enhanced on coating with gold nanoparticle against vancomycin resistant enterococci (VRE) [72]. The coating of aminoglycosidic antibiotics with gold nanoparticles has an antibacterial effect on a range of Gram-positive and Gram-negative bacteria [73-74]. Cefaclor (a second-generation lactam antibiotic) reduced gold nanoparticles have potent antimicrobial activity on both Grampositive (S. aureus) and Gram-negative bacteria (E. coli) compared to cefaclor and gold nanoparticles alone. Cefaclor inhibits the synthesis of the peptidoglycan layer, making cell walls porous. Further, the gold nanoparticles generate holes in the cell wall, resulting in the leakage of cell contents and cell death. It is also possible that gold nanoparticles bind to the DNA of bacteria and inhibit the uncoiling and transcription of DNA [75].The Au nanoparticles can be used to coat a wide variety of surfaces for instance implants, fabrics for treatment of wounds and glass surfaces to maintain hygienic conditions in the home, in hospitals and other places [76]. 3.1.3 Magnesium oxide nanoparticles Highly ionic nanoparticulate metal oxides can be prepared with extremely high surface areas and unusual crystal morphologies having numerous edge/corner and reactive surface sites [77]. Magnesium oxide (MgO) prepared through an aerogel procedure (AP-MgO) yields square and polyhedral shaped nanoparticles with diameters varying slightly around 4 nm, arranged in an extensive porous structure with considerable pore volume [78]. An interesting property of AP-MgO nanoparticles is their ability to adsorb and retain for a long time (in the order of months) significant amounts of elemental chlorine and bromine [79]. The AP-MgO/X2 nanoparticles exhibited biocidal activity against certain vegetative Gram-positive bacteria, Gram-negative bacteria and the spores [80]. AP-MgO nanoparticles are found to possess many properties that are desirable for a potent disinfectant [81]. Because of their high surface area and enhanced surface reactivity, the nanocrystals adsorb and carry a high load of active halogens. Their extremely small size allows many particles to cover the bacteria cells to a high extent and bring halogen in an active form in high concentration in proximity to the cell [80]. Standard bacteriological tests have shown excellent activity against E.coli

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and Bacillus megaterium and a good activity against spores of Bacillus subtilis [81]. The bioactivity of AP-MgO/X2 nanoparticles is due to the positive charge they have in water suspension, opposite to those of the bacteria and spore cells, which enhances the total bactericidal effect. Confocal microscopy studies have shown that in water suspension the opposite charge brings the bacteria and nanoparticles together in aggregates composed of both AP-MgO nanoparticles and bacteria. Atomic force microscopy and electron microscopy studies demonstrate that halogenated magnesium oxide has a very strong influence on microorganisms and their membranes in particular. Overall, the halogen such as chlorine and bromine treated MgO nanoparticles have a stronger and faster effect on the killing action of both bacteria and spores [81]. 3.1.4 Copper oxide nanoparticles Copper oxide (CuO) is a semiconducting compound with a monoclinic structure. It is the simplest member of the family of copper compounds and exhibits a range of potentially useful physical properties such as high temperature, superconductivity, electron correlation effects and spin dynamics. Therefore, it finds a wide application [82-83]. CuO crystal also has photocatalytic or photovoltaic properties and photoconductive functionalities [84]. There is limited information available about the antimicrobial activity of nano CuO. As CuO is cheaper than silver, easily mixes with polymers and relatively stable in terms of both chemical and physical properties, it finds a wide application [85]. It is suggested that highly ionic nanoparticulate metal oxides, such as CuO, may find potential application as antimicrobial agents as they can be prepared with extremely high surface areas and unusual crystal morphologies [77]. CuO nanoparticles were effective in killing a range of bacterial pathogens involved in hospital-acquired infections. But a high concentration of nano CuO is required to achieve a bactericidal effect [86]. It has been suggested that the reduced amount of negatively charged peptidoglycans makes Gram-negative bacteria such as Pseudomonas aeruginosa and Proteus spp. less susceptible to such positively charged antimicrobials. However, in the timekill experiments the Gram-negative strains showed a greater susceptibility to nano CuO combined nano Ag. Studies have been conducted to assess the potential of nano CuO embedded in a range of polymer materials. A lower contact-killing ability was observed in comparison with release killing ability against MRSA strains. This suggests that a release of ions into the local environment is required for optimal antimicrobial activity [86-87]. Copper nanoparticles have a high antimicrobial activity against B. subtilis. This may be attributed to greater abundance of amines and carboxyl groups on cell surface of B. subtilis and greater affinity of copper towards these groups. Copper ions released may also interact with DNA molecules and intercalate with nucleic acid strands. Copper ions inside bacterial cells also disrupt biochemical processes [88]. The exact mechanism behind bactericidal effect of copper nanoparticles is not clear. 3.1.5 Aluminium nanoparticles Aluminum oxide NPs have wide-range applications in industrial and personal care products. The growth-inhibitory effect of alumina NPs over a wide concentration range (101000 g/mL) on Escherichia coli have been studied [89]. Fourier transforminfrared studies have shown differences in structure between nanoparticles treated and untreated cells. Alumina nanoparticle have exhibited a mild growth-inhibitory effect, only at very high concentrations. This is attributed to surface charge interactions between the particles and cells. It is possible that the free-radical scavenging properties of the particles might have prevented cell wall disruption and drastic antimicrobial action [89]. Alumina is thermodynamically stable over a wide temperature range and has a corundum-like structure, with oxygen atoms adopting hexagonal close packing and Al3+ ions filling two thirds of the octahedral sites in the lattice [90]. The alumina nanoparticles carry a positive charge on its surface at near-neutral pH. The electrostatic interaction between the negatively charged E. coli cells and the particles resulted in the adhesion of nanoparticles on the bacterial surfaces [91]. The adhesion increased with increase in concentration of the particles in the suspension, a negative effect on growth was observed with respect to concentration. This electrostatic interaction between bacteria and particle surface, along with hydrophobic interactions and polymer bridging, may be responsible for the phenomenon of bacterial adhesion onto the particles. The antimicrobial property of these metal oxides is attributed to the generation of reactive oxygen species (ROS) which causes disruption of cell wall and subsequntly cell death [88]. But alumina NPs may act as free radical scavengers. These NPs are able to rescue cells from oxidative stress-induced cell death in a manner that appears to be dependent upon the structure of the particle but independent of its size within the range of 61000 nm [92]. 3.1.6 Titanium dioxide nanoparticles The inhibitory activity of TiO2 is due to the photocatalytic generation of strong oxidizing power when illuminated with UV light at wavelength of less than 385 nm [93-95]. TiO2 particles catalyze the killing of bacteria on illumination by near-UV light. The generation of active free hydroxyl radicals (_OH) by photoexcited TiO2 particles is probably responsible for the antibacterial activity [96-98]. The antimicrobial effect of TiO2 photocatalyst on Escherichia coli in water and its photocatalytic activity against fungi and bacteria has been demonstrated [99-102].

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There are also studies on bactericidal activity of nitrogen-doped metal oxide nanocatalysts on E. coli biofilms and on the photocatalytic oxidation of biofilm components on TiO2- coated surfaces [100]. In conclusion, the use of TiO2 photocatalysts as alternative means of self-disinfecting contaminated surfaces by further development may provide potent disinfecting solutions for prevention of biofilm formation. TiO2 photocatalysts can be used as effective biofilm disinfectant in food processing industries [103-104]. Suspensions containing TiO2 are effective at killing Escherichia coli. This has led to the development of photocatalytic methods for the killing of bacteria and viruses using TiO2 in aqueous media [105-106]. It has been suggested that nanostructured TiO2 on UV irradiation can be used as an effective way to reduce the disinfection time, eliminating pathogenic microorganisms in food contact surfaces and enhance food safety [93]. The major disadvantage of using TiO2 is that UV light is required to activate the photocatalyst and initiate the killing of the bacteria and viruses. In recent years, visible light absorbing photocatalysts with Ag/AgBr/TiO2 has proved to be successful at killing S. aureus and E. coli [107-108]. 3.1.7 Zinc oxide nanoparticles Among the various metal oxides studied for their antibacterial activity, zinc oxide nanoparticles have been found to be highly toxic. Moreover, their stability under harsh processing conditions and relatively low toxicity combined with the potent antimicrobial properties favours their application as antimicrobials [77]. Many studies have shown that some NPs made of metal oxides, such as ZnO NP, have selective toxicity to bacteria and only exhibit minimal effect on human cells, which recommend their prospective uses in agricultural and food industries [109-112]. The antimicrobial activity of zinc oxide nanoparticles have been studied against the food related bacteria Bacillus subtilis, Escherichia coli and Pseudomonas fluorescens [113]. ZnO NP could potentially be used as an effective antibacterial agent to protect agricultural and food safety from foodborne pathogens, especially E. coli O157:H7 [112]. ZnO NPs possess antimicrobial activities against Listeria monocytogenes, Salmonella enteritidis and E. coli O157:H7 in culture media [113]. There are also other studies confirming the strong antimicrobial activity of ZnO nanoparticles wherein the nanoparticles could completely lyse the food-borne bacteria Salmonella typhimurium and Staphylococcus aureus [114]. In another study, ZnO nanoparticles (12 nm) inhibited the growth of E. coli by disintegrating the cell membrane and increasing the membrane permeability [115]. The above findings suggest that ZnO nanoparticles can find applications in food systems and can be used to inhibit growth of pathogenic bacteria. There are several mechanisms which have been proposed to explain the antibacterial activity of ZnO nanoparticles. The generation of hydrogen peroxide from the surface of ZnO is considered as an effective mean for the inhibition of bacterial growth [116]. It is presumed that with decreasing particle size, the number of ZnO powder particles per unit volume of powder slurry increases resulting in increased surface area and increased generation of hydrogen peroxide. Another possible mechanism for ZnO antibacterial activity is the release of Zn2+ ions which can the damage cell membrane and interact with intracellular contents [109]. 3.2 Antiviral studies on nanoparticles The antibacterial property of metal nanoparticles has been extensively explored. But the anti-viral properties of metal nanoparticles remain an undeveloped area [117]. The diseases caused by viruses present challenging problems with worldwide social and economic implications. Developing antiviral drugs which can target the virus and maintaining host cell viability is challenging [118]. Metal NPs have been proposed as anti-viral systems taking advantage of the core material and/or the ligands shell [119]. Silver nanoparticles have proven to exert antiviral activity against HIV-1 at non-cytotoxic concentrations [120]. Using a number of in vitro assays, the mode of antiviral action of silver nanoparticles against HIV-1 was elucidated. It was revealed that silver nanoparticles exert anti-HIV activity at an early stage of viral replication, most likely as a virucidal agent or as an inhibitor of viral entry. Silver nanoparticles prevent CD4-dependent virion binding, fusion, and infectivity, acting as an effective virucidal agent against both cell-free virus and cell-associated virus. Besides, silver nanoparticles inhibit post-entry stages of the HIV-1 life cycle. It can be concluded that these properties make them a broad-spectrum agent not prone to inducing resistance that could be used preventively against a wide variety of circulating HIV-1 strains [120]. Gold nanoparticles have also been explored for their anti-HIV activity [119]. The gold nanoparticles coated with multiple copies of an amphiphilic sulfate-ended ligand bind the HIV envelope glycoprotein gp120 and inhibit the HIV infection of T-cells at nanomolar concentrations in vitro. Nanoparticle-based presentation of multiple ligands creates a high local concentration of binding molecules which can assist the targeted biological interaction. It is suggested that the multivalent gold nanoparticles coated with sulfated ligands can be considered a novel alternative to the known antiHIV systems for targeting the adsorption/fusion process of the virus infection. Depending on the ligand density, these NPs can bind gp120 with high affinity [119]. The possibility of simultaneously tailoring on the same gold nanoplatform both sulfated ligands and other active molecules which target different stages of the HIV life cycle makes gold

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nanoparticles an appealing scaffold which can contribute to the development of new multifunctional anti-HIV systems [121]. The use of gold nanoparticles with different anionic groups to inhibit influenza has also been reported [122]. The anionic gold nanoparticles have shown effective inhibition properties against several influenza strains. The primary mechanism of inhibition was due to the blocking of viral attachment to cell surface. The variation in the degree of inhibition with the anionic groups indicated that in the antiviral activity of the nanoparticles, the charge density and the functional group itself has a role to play. The end groups present on the surface of the anionic gold functionalized groups may interact with the viruses by multivalent bonds. The small size of the gold nanoparticles (Au-NPs) enables them to enter the cell through the endosome vesicle allowing them to possibly interfere with the fusion step as well [123]. Several publications demonstrated low or no Au-NPs cytotoxicity, and their toxicity if present is dependent on the particles surface charge, size and shape [124]. The NPs composed of an Au core and mercaptoethanesulfonate (MES) molecules bonded to its surface through the thiol group exhibited no cytotoxicity and inhibited different influenza virus types and strains (including the recent pandemic swine influenza A (H1N1) strain), indicating that the interaction is not limited to a specific strain. This property is crucial for antiviral compounds, which need to be unaffected by random viral genetic mutations. There are also studies on the inhibition of Herpes simplex virus Type 1 infection by silver nanoparticles capped with mercaptoethane sulfonate [118]. These nanoparticles target the virus by competing for its binding to cellular heparan sulfate through their sulfonate end groups. This results in the blockage of viral entry into the cell and prevention of subsequent infection. Effective inhibition of HSV-1 infection in cell culture by the capped nanoparticles has been demonstrated. There are no toxic effects of these nanoparticles on host cells. The capped nanoparticles can serve as useful topical agents for the prevention of infections with pathogens dependent on heparan sulfate for entry. There are also several studies which report the antiviral activity of silver nanoparticles against the hepatitis B virus, respiratory syncytial virus, HIV 1 and monkeypox virus [125-128]. These studies suggest that metal nanoparticles with functionalized groups have enhanced antiviral activity and may possibly help in combating viral infections. 3.3 Antifungal activity of metal nanoparticles In the recent years, severe fungal infections have significantly contributed to the increasing morbidity and mortality of immunocompromised patients who need intensive treatment including broad-spectrum antibiotic therapy [129]. There are limited studies on the antifungal activity of metal nanoparticles. The fungistatic and fungicidal effects of the silver NPs against selected pathogenic yeasts causing invasive life-threatening fungal infections in intensive care patients has been studied. Silver NPs exhibit high antifungal activity against pathogenic Candida spp. at a concentration of around 1 mg/L of Ag. Antifungal activity of the silver NPs is comparable with those of ionic silver [130]. The antifungal effects of spherical silver nanoparticles have been investigated against dermatophytes [131]. The Nano-Ag exhibited potent activity against clinical isolates and ATCC strains of Trichophyton mentagrophytes and Candida species (IC80, 1-7 g/ml). The activity of nano-Ag was comparable to that of amphotericin B and fluconazole. The antifungal activity of nano-Ag is attributed to its effects on the mycelia [131]. There are also reports of the application of nanosilver having antifungal activity in biostabilization of footwear materials, wherein, 1% solution inhibited the growth of the majority of yeast-like fungal and mold strains [132]. The mode of action of nano-Ag on fungi is by targeting the yeast cell membranes and disrupting membrane potential. The transmission electron microscopy analysis has revealed that the interaction between nano-Ag and the membrane structure of C. albicans cells during nano-Ag exposure results in changes in the membranes of C. albicans, which can be observed as the pits on the membrane surfaces. The formation of pores subsequently leads to cell death [133].

4. Application of nanoparticles
It is evident that the metal based nanoparticles constitute an effective antimicrobial agent against common pathogenic microorganisms. Therefore, some of the nanoparticles such as silver, titanium dioxide and zinc oxide are receiving considerable attention as antimicrobials and additives in consumer, health-related and industrial products [44]. As silver nanoparticles have a broad spectrum antimicrobial activity against several pathogens they are increasingly incorporated into various matrices to extend their utility in materials and biomedical applications [50]. They are used as additives in health related products such as bandages, catheters, and other materials to prevent infection, particularly during the healing of wounds and burns. An antibacterial Ag/Na carboxymethyl cotton burn dressing by the partial cation exchange of sodium with silver has been developed and these can find applications in surgical dressings. They are currently being added to many common household products such as bedding, washers, water purification systems, tooth paste, shampoo, fabrics, deodorants, filters, paints, kitchen utensils, toys, and humidifiers to impart antimicrobial properties [55]. Nanoparticles of titanium dioxide are used in cosmetics, filters that exhibit strong germicidal properties and remove odors, and in conjunction with silver as an antimicrobial agent. Moreover, due to the photocatalytic activity, it has been

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used in waste water treatment. It is considered non-toxic and has been approved by the American Food and Drug Administration (FDA) for use in human food, drugs, cosmetics and food contact materials [134]. Nowadays titanium dioxide nanoparticles are finding wide application as a self-cleaning and self-disinfecting material for surface coatings in many applications and in food industries for disinfecting equipments [135]. Zinc oxide (ZnO) and copper oxide nanomaterials due to their antimicrobial property are being incorporated into a variety of medical and skin coatings. ZnO nanoparticles are used in the wallpapers in hospitals as antimicrobials. ZnO powder is an active ingredient for dermatological applications in creams, lotions and ointments on account of its antibacterial properties [90].

5. Toxicity of nanoparticles
It is evident from the above studies that metal nanoparticles due to their unique physico-chemical and biological properties have far reaching industrial and medical applications. But there is a dearth of knowledge about the effect of the prolonged exposures to nanoparticle on human health and environment. The implication of nanoparticles on health and environment needs to be assessed completely before their large-scale production and application in various fields [136-138]. Studies conducted on the NP-induced toxicity have revealed that the metal-based nanoparticles can affect the biological behavior at the organ, tissue, cellular, subcellular, and protein levels. The size of the nanoparticles is small and these can easily access the skin, lungs, and brain and cause adverse affects [139-142]. For example, exposure of metal based nanoparticles to human lung epithelical cells leads to the generation of reactive oxygen species and result in oxidative stress and cellular damage. The toxicity of nanoparticles can be assessed by a number of in vitro and in vivo studies. For example, the toxic effects of nanoparticles can be carried out using zebrafish as a model due to its fast development and transparent body structure. Cell culture based assays are used as a pre-screening tool to understand the biological effects of nanoparticles. However, along with the in vitro assays it is necessary to confirm the in vivo biological activities of nanoparticles in animal models to study the suitability of their application [143-144]. There is an increasing use of microarray and real-time reverse transcription polymerase chain reaction for gene expression analyses as these are very sensitive and reliable methods to assess the changes in the expression levels of thousands of genes simultaneously under a wide variety of experimental conditions [145]. It is evident that metal based nanoparticles due to their biological and physiochemical properties are promising as antimicrobials and therapeutic agents. They can be used to address a number of challenges in the field of nanomedicine. But it must be remembered that they can also possibly cause adverse biological effects at the cellular and subcellular levels. Therefore, after the cytotoxicity and clinical studies the nanoparticles can find immense application as antimicrobials in the consumer and industrial products.
Table 1 Antimicrobial activity of metal based nanoparticles.

Properties Antibacterial

Mechanism of action Interaction with phosphorus moieties in DNA, resulting in inactivation of DNA replication. Reacts with sulfur-containing proteins, leading to the inhibition of enzyme functions.

Examples of nanoparticles Silver nanoparticles have inhibitory activity against E. coli, B. subtilis, S. aureus, methicillin-resistant coagulase-negative staphylococci, vancomycin-resistant Enterococcus faecium, ESBL-positive K. pneumonia, S. typhi, Vibri cholera [29, 30, 32, 45, 52]. Gold nanoparticles have antibacterial activity against MRSA, VRE, E. coli, Pseudomonas aeruginosa [65-66, 72]. MgO nanoparticles have excellent against E. coli, B.subtilis, B.megaterium.[80,81] CuO strongly inhibits B.subtilis [86-88]. Aluminium oxide nanoparticles have growth inhibitory effect on E. coli [91]. TiO2 nanoparticls are effective in killing E. coli, S.aureus, Listeria monocytogenes [99-102, 107,108]. ZnO nanoparticles inhibit food-borne bacteria E. coli 0157:H7,B.subtilis, Pseudomonas fluorescens, L. monocytogenes, Salmonella enteritidis, S. aureus, S. typhimurium [113-115]. Gold nanoparticles have anti-HIV activity and inhibit several strains of influenza virus [119-120]. Silver nanoparticles inhibit HIV-1, Influenza virus, Herpes Simplex virus, Respiratory syncytial virus, Monkey pox virus [118,120, 125-128]. Silver nanoparticles have fungicidal and fungistatic effects on the dermatophytes Trichophyton mentagrophytes and Candida species [130-133].

Antiviral

Blocking of viral attachment to cell surface.

Antifungal

Disruption of cell membrane.

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Science against microbial pathogens: communicating current research and technological advances _______________________________________________________________________________ A. Mndez-Vilas (Ed.)

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