WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Poonam World Journal of Pharmacy and Pharmaceutical Sciences SJIF Impact Factor 2.786 Volume 4, Issue 04, 652-677. Review Article ISSN 2278 – 4357 A REVIEW ON SYNTHESIS AND THEIR ANTIBACTERIAL ACTIVITY OF SILVER AND SELENIUM NANOPARTICLES AGAINST BIOFILM FORMING STAPHYLOCOCCUS AUREUS Poonam Verma* School of Biotechnology, IFTM University, Moradabad, Uttar Pradesh, India. ABSTRACT Article Received on 29 Jan 2015, Antibacterial agents are very important in the textile industry, water Revised on 22 Feb 2015, Accepted on 18 March 2015 disinfection, medicine, and food packaging. Organic compounds used for disinfection have some disadvantages, including toxicity to the human body; therefore, the interest in inorganic disinfectants such as *Correspondence for Author metal oxide nanoparticles (NPs) is increasing. This review focuses on Poonam Verma the Preparation and their potential with good antimicrobial activity of School of Biotechnology, Ag-NPs and Se-NPs against biofilm forming S. aureus. Such improved IFTM University, antibacterial agents locally destroy bacteria, without being toxic to the Moradabad, Uttar surrounding tissue. We also provide an overview of opportunities and Pradesh, India. risks of using NPs as antibacterial agents. In particular, we discuss the role of Ag-NPs and Se-NPs materials. Several manufactured nanoparticles- particles with one dimension less than 100 nm are increasingly used in consumer products. At nano size range, the properties of materials differ substantially from bulk materials of the same composition, mostly due to the increased specific surface area and reactivity, which may lead to increased bioavailability and toxicity. Thus, for the assessment of sustainability of nanotechnologies, methods of manufacturing Nanoparticles, properties have to be studied. The formation of nanoparticle and physiochemical parameters such as pH, monomer concentration, ionic strength as well as surface charge, particle size and molecular weight are important for drug delivery. Further, these nanoparticles have the capability to reverse multidrug resistance a major problem in chemotherapy. Well-established therapies commonly employed in cancer treatment include surgery, Chemotherapy, immunotherapy, and radiotherapy. The silver nanoparticles might be involved in neutralizing these adhesive substances, thus preventing biofilm formation. Selenium is also one of essential trace elements in the human body and has great importance in nourishment and medicine. Medical www.wjpps.com Vol 4, Issue 04, 2015. 652 Poonam World Journal of Pharmacy and Pharmaceutical Sciences diagnostic field also developed to use the selenium nanoparticles and also studies on the increase efficiency of glutathione peroxidase and thioredosin reductase. KEYWORDS: Nanoparticles, Chemotherapy, Immunotherapy, Radiotherapy, Antibacterial agents, Disinfection. INTRODUCTION 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.[1] 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.[2] 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.[3] 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.[4] 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. 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.[5] A microbial infection either from bacteria and/or yeast can lead to high morbidity and even mortality of patients worldwide. Microbial adhesion to medical devices surfaces is considered the base of the pathogenic mechanism, so a continuous concern to the medical community is the high rate of infection of biomaterials which are inserted into or in contact with the human body. As an example on the orthopaedic field, the percentage of implant failure due to infection is approximately 1.5–2.5% of all implants (3000–6000 incidents per year).[6] The failure of such devices relies on the ability of microorganisms to adhere to submerged www.wjpps.com Vol 4, Issue 04, 2015. 653 Poonam World Journal of Pharmacy and Pharmaceutical Sciences surfaces and produce extracellular substances that facilitate adhesion and provide a structural matrix, forming multicellular communities the biofilms.[7] Drug delivery nanocarriers systems, such as liposomes[8] and polymer-based[9] carriers have also arisen as appealing methods with a great potential in the treatment of biofilm infections, due to several factors especially good biocompatibility and ample range and extent of drugs that they can carry. Another important factor is the protection provided by the encapsulation of the drug in the biological milieu, decreasing toxicity and allowing the drug to reach the specific site. With the emergence and increase of microbial organisms resistant to multiple antibiotics, and the continuing emphasis on health-care costs, many researchers have tried to develop new, effective antimicrobial reagents free of resistance and cost. Such problems and needs have led to the resurgence in the use of Ag-based antiseptics that may be linked to broad-spectrum activity and far lower propensity to induce microbial resistance than antibiotics.[10] Silver nanocrystals, mostly hydrosols are one of the most attractive inorganic material not only because of its tremendous applications in photography[11], catalysis [12] , biosensor[13], biomolecular detection[14], diagnostics[15], and particularly antimicrobial [16, 17, 18] activities but also because of its environmentally benign nature.[19,20,21,22] Synthesis of different morphologies of advanced silver nanomaterials (nanotubes, nanowires, nano cubes, nanorods, and nanosheets) has been the subject of a large number of researchers in many laboratories.[23, 24, 25, 26, 27] A number of methods were used in the past for the synthesis of silver nanoparticles for example, reduction in solutions [28], radiation assisted[29] chemical and photoreduction in reverse micelles[30], thermal decomposition of silver compounds[31] and recently via bio- or green-synthesis route.[17, 26, 32] In general, certain selenium compounds are catalytic and produce ROS by their interaction with thiols, such as reduced glutathione, forming the glutathione selenide anion, GSSe.¯ [33] Reactive Oxygen Species (ROS), including superoxide radical, hydrogen peroxide, and hydroxide radical, can cause cellular damages such as DNA oxidation, lipid peroxidation, and protein oxidation.[34, 35, 36] Certain anti-tumor drugs, such as Tallysomycin, Bleomycin, Adriamycin, and anticancer drugs, such as quinines, produced ROS which kills tumor cells.[37, 38, 39, 40] Moreover, ROS has been shown to kill different bacteria such as Staphylococcus epidermidis, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, and Escherichia coli in vitro[41, Chromobacterium violaceum in vivo.[46, www.wjpps.com 47] 42, 43, 44, 45] and Burkholderia cepacia and The paradox of selenium (Se) is that it is both Vol 4, Issue 04, 2015. 654 Poonam World Journal of Pharmacy and Pharmaceutical Sciences essential and toxic. According to the World Health Organization (WHO), a recommended daily dietary selenium intake is 40 μg Se/day.[48] However, selenium is not toxic at high level (3200 μg/day).[49] Therefore, in this review, we focus on the Synthesis of metallic Silver and Selenium nanoparticles as well as of Silver and Selenium metallic nanoparticles as potential antimicrobials and the possible mechanism of their inhibitory actions on biofilm forming S. aureus. The increasing application of nanoparticles as antimicrobials in biomedical associated with these particles will also be reviewed. SYNTHESIS OF SILVER AND SELENIUM NANOPARTICLES BY CHEMICAL METHODS SILVER NANOPARTICLES SYNTHESIS: The silver colloid was prepared by using chemical reduction method. All solutions of reacting materials were prepared in distilled water. In typical experiment 0.0849 gm of AgNO3 was dissolved in 500 ml distilled water, and then the solution was heated to boiling. Then 1 gm of tri sodium citrate was dissolved in 100 ml distilled water and 5 ml of trisodium citrate were added to 500 ml of AgNO3 after boiling (drop by drop). During the process, the solution was mixed vigorously. The solution was left on hot plate for 2 hours at 90°C for heating only, then it was cooled to room temperature, the colour was reddish green.[50] Fig 1: Reddish green Ag-NPs obtained Fig 2: SEM image of Ag-NPs Silver nanoparticles were synthesized by chemical reduction method. Silver nitrate was taken as the metal ancestor and hydrazine hydrate as a reducing agent. The development of the silver nanoparticles was monitored using UV-Vis absorption spectroscopy. The UV-Vis spectroscopy revealed the formation of silver nanoparticles by exhibiting the typical surface www.wjpps.com Vol 4, Issue 04, 2015. 655 Poonam World Journal of Pharmacy and Pharmaceutical Sciences plasmon absorption maximum at 418-420 nm from the UV–Vis spectrum. The average size and morphology of silver nanoparticles were determined by transmission electron microscopy (TEM). TEM photographs indicate that the nano powders consist of well dispersed agglomerates of grains with a narrow size distribution (40 and 60 nm), whereas the radius of the singly particles are between 10 and 20 nm. The synthesized nanoparticles have been structurally characterized by X-ray diffraction and transmission high-energy electron diffraction (HEED). The peaks in the XRD pattern are in excellent agreement with the standard values of the face-centered-cubic form of metallic silver and no peaks of other impurity crystalline phases were detected. Additionally, the antibacterial activity of the nano particulars dispersion was considered by Kirby-Bauer method. The nanoparticles of silver showed high antimicrobial and bactericidal activity against gram positive bacteria such as Escherichia Coli, Pseudimonas aureginosa and Staphylococcus aureus which is a highly methicillin resistant strain.[51] Flat sheet porous polysulfone–silver nano combination membranes were synthesized by the wet phase inversion process. The effects of casting mixture composition and nanoparticle incorporation route on the morphological and disjointing properties of prepared membranes were studied by comparing nano composites of different preparations with silver-free controls. Silver nanoparticles were either synthesized ex situ and then added to the casting solution as an organosol or produced in the casting solution via in situ reduction of ionic silver by the polymer solvent. Nano composite membranes of three types differing in skin porosity and macro void structure were organized. The structure and properties of nano composites were interpreted in terms of the coupling between the processes of nanoparticle development and gelling of the polymer-rich phase during phase inversion. Larger nanoparticles preferentially situated in the skin layer were experimental in composites prepared via the ex situ method while in situ reduction of silver led to formation of smaller nanoparticles homogeneously distributed along the membrane cross-section. In some cases, incorporation of nanoscale silver formed ex situ resulted in macro void widening and an order of magnitude decrease in hydraulic resistance accompanied by only a moderate decrease in rejection. The accessibility of the silver nanoparticles embedded in the membrane was quantitatively assessed by the degree of the growth inhibition of a membrane biofilm due to the ionic silver released by the nano composites and was found to depend on the process of silver incorporation.[52] www.wjpps.com Vol 4, Issue 04, 2015. 656 Poonam World Journal of Pharmacy and Pharmaceutical Sciences The development of green experimental processes for the synthesis of nanoparticles is a need in the field of nanotechnology. The synthesis of silver nanoparticles was achieved using Bacillus cereus supernatant and 1 mM silver nitrate. 100 mM glucose was initiated to quicken the rate of reaction of silver nanoparticles synthesis. UV-visible spectrophotometric investigation was carried out to assess the synthesis of silver nanoparticles. The synthesized silver nanoparticles were extra characterized by using Nanoparticle Tracking Analyzer (NTA), Transmission Electron Microscope and Energy Dispersive X-ray spectra. These silver nanoparticles showed enhanced quorum quenching activity against Staphylococcus aureus biofilm and prevention of biofilm formation which can be seen under inverted microscope (40 X). The synergistic effect of silver nanoparticles along by antibiotics in biofilm quenching was established to be effective. In the near future, silver nanoparticles could be used in the treatment of infections caused by vastly antibiotic resistant biofilm.[53] SELENIUM NANOPARTICLES SYNTHESIS- Selenium nanoparticles (SeNPs) were synthesized by the reduction of sodium selenite by glutathione (reduced form) and stabilized by bovine serum album in (BSA). Specifically, 3 mL of 25 mM Na2SeO3, 3 mL of 100mM GSH, and 0 .15 g BSA were added to 9 mL of double distilled water in a sterile cabinet. All solutions were made in a sterile environment by using a sterile cabinet and double distilled water. After mixing the reactant solution, 1 M NaOH was added to bring the pH of the solution to the alkaline regimen. Selenium nanoparticles was formed immediately following the addition of NaOH as visualized by a color change of the reactant solution from clear white to clear red. Selenium nanoparticles were then collected by centrifuging the solution at 13,000 rpm sterilized by ultra-violet light exposure, and re-suspended in sterile double distilled water five times before use in bacteria experiments.[54] Fig 3: Red Se-NPs obtained www.wjpps.com Fig 4: SEM image of Se-NPs Vol 4, Issue 04, 2015. 657 Poonam World Journal of Pharmacy and Pharmaceutical Sciences In this authors study, selenium was originated from an industrial dust. After the distillation method, the researchers were able to prepare selenium micro powder. The objective of this study was to prepare selenium nano particles through water solution phase method from the micro powders. The results showed that selenium micronized powder as well as nano particles had high purity. The obtained nanoparticles were tube shaped with 20 nm size.[55] In the current lessons, selenium (Se) nanoclusters were developed through various nucleations on titanium (Ti) surfaces, a common orthopedic insert material. Common healthy osteoblasts (bone-forming cells) and cancerous osteoblasts (osteosarcoma) were cultivated on the Se-doped surfaces having 3 dissimilar coating densities. For the first time, it is shown that substrates with Se nanoclusters promote normal osteoblast proliferation and inhibit cancerous osteoblast growth in both detach (mono-culture) and sub-culture experiment. This study suggests that Se surface nanoclusters can be appropriately engineered to reduce bone cancer growth while concurrently promoting the growth of normal bone tissue.[56] The bacteria exhibited important patience to selenite (SeO3--) up to 100 mM conc. with an EC50 value of 140 mM. The spent medium (culture supernatant) contains the potential of reducing soluble and colorless SeO3-- to insoluble red elemental selenium (Seo) at 37°C. description of red Se° manufactured by use of UV-Vis spectroscopy, X-ray diffraction (XRD), atomic force microscopy (AFM) and transmission electron microscopy (TEM) with energy dispersive X-ray spectrum (EDX) analysis revealed the presence of stable, predominantly monodispersed and spherical selenium nanoparticles (Se-NPs) of an average size of 21 nm. Most likely, the metabolite phenazine-1-carboxylic acid (PCA) free by Pseudomonas aeruginosa strain in culture supernatant alongside with the known redox agents like NADH and NADH dependent reductases are dependable for biomimetic reduction of SeO3-- to Se° nanospheres. Based on the bioreduction of a colorless solution of SeO3-- to elemental red Se0, a high throughput colorimetric bioassay (Se-Assay) was developed for parallel revealing and quantification of nanoparticles (NPs) cytotoxicity in a 96 well plate. Thus, it has been accomplished that the reducing power of the culture supernatant of Pseudomonas aeruginosa strain could be efficiently demoralized for developing a troublefree and environmental friendly process of Se-NPs production. The results elucidated that the red colored Se° nanospheres may serve as a biosensor for nanotoxicity consideration, contemplating the inhibition of SeO3-- bioreduction process in NPs treated bacterial cell society supernatant, as a toxicity end point.[57] www.wjpps.com Vol 4, Issue 04, 2015. 658 Poonam World Journal of Pharmacy and Pharmaceutical Sciences ANTIMICROBIAL EFFECTS OF Ag-NPs The present study was undertaken to estimate pharmacological inhibition of biofilm of clinical isolate of Staphylococcus aureus on silver nanoparticles coated catheter under in vitro condition and the synergistic effect of nanoparticles with antibiotics was studied. Silver nanoparticles synthesized by chemical reduction process were coated on the catheter adopting simple dispersion process. Initially biofilm inhibitory effect, authors was studied with spectrophotometric method and the biochemical composition of biofilm matrix mainly total carbohydrates and protein studied. Silver nanoparticles synthesized by chemical reduction of silver nitrate with sodium borohydride were coated on the catheter was characterized by surface topography of catheter by scanning electron microscopy which reveals complete dispersion of the nanoparticles on the fibre surface of the catheter and the size, shape of the particles shows uniform spherical particles with the size of 40-60 nm. Distinct effect of biofilm inhibition was recorded in the nanoparticles coated catheter and maximum inhibition was observed during 24th hour of incubation Surface topography of nanoparticles and nanoparticles with antibiotics coated catheter with scanning electron microscopy reveals the entire degeneration of the biofilm whereas biofilm matrix biochemical composition primarily total carbohydrates and total protein was highly reduced. Biofilm inhibition rate and reduction of biofilm matrix biochemical composition was increased in nanoparticles with all the tested antibiotics action which suggests the synergistic effect.[58] An AgNPs solution was prepared by chemical reduction, characterized, and tested against Candida glabrata, Candida tropicalis, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus (MRSA). Minimum inhibitory (MICs) and minimum fungicidal/bactericidal concentrations (MFC/MBC) were determined on planktonic cells. Also, total biofilm mass was determined by crystal violet (CV) staining and morphological changes by scanning electron microscope (SEM). MICs for C. glabrata, C. tropicalis, S. aureus, and MRSA were 15.63, 3.91, 1.95, and 1.95 𝜇g/mL, respectively. MFC for C. glabrata was 62.5 𝜇g/mL and for C. tropicalis 15.63 𝜇g/mL. The same MBC (3.91 𝜇g/mL) was observed for S. aureus and MRSA. CV assay showed that the AgNPs (1000 𝜇g/mL) promoted reductions in biofilm mass of ∼60% for C. glabrata and ∼35% for C. tropicalis. A reduction of ∼20% in C. tropicalis biomass was also observed at the concentration 3.91 𝜇g/mL. No significant effect on total biomass was established for S. aureus and MRSA. SEM images exposed that C. glabrata and C. tropicalis biofilm cells, exposed to the AgNPs (1000 𝜇g/mL), had an irregular and shriveled appearance. AgNPs solution exhibited considerable www.wjpps.com Vol 4, Issue 04, 2015. 659 Poonam World Journal of Pharmacy and Pharmaceutical Sciences antimicrobial activity against significant fungal and bacterial pathogens, associated with several oral and systemic diseases, and has potential as an antimicrobial agent.[59] Silver nanoparticles (nano-Ags), which have familiar antimicrobial properties, are used broadly in various medical and general applications. In this study, the combination effects between nano-Ags and the conventional antibiotics ampicillin, chloramphenicol and kanamycin against various pathogenic bacteria were investigated. The MIC and fractional inhibitory concentration index (FICI) were determined to confirm antibacterial susceptibility and synergistic effects. The results showed that nano-Ags possessed antibacterial effects and synergistic activities. The antibiofilm activities of nano-Ags alone or in combination with antibiotics were also investigated. Formation of biofilm is associated with resistance to antimicrobial agents and chronic bacterial infections. The results indicated that nano-Ags also had antibiofilm activities. To understand these effects of nano-Ags, an ATPase inhibitor assay, permeability assay and hydroxyl radical assay were conducted. The antibacterial activity of nano-Ags was influenced by ATP associated metabolism rather than by the permeability of the outer membrane. Furthermore, nano- Ags generated hydroxyl radicals, a greatly reactive oxygen species induced by bactericidal agents. It was concluded that nanoAg have prospective as a combination therapeutic agent for the treatment of infectious diseases by bacteria.[60] Emerging infectious diseases and amplify in incidence of drug resistance among pathogenic bacteria have made the search for new antimicrobials inevitable. In the current condition, one of the mainly promising and novel therapeutic agents is 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 single and well defined physical and chemical properties which can be manipulated suitably for preferred 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. In this review authors focuses on the properties of dissimilar www.wjpps.com Vol 4, Issue 04, 2015. 660 Poonam World Journal of Pharmacy and Pharmaceutical Sciences 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 troubles related to toxicity of nanoparticles will be addressed in brief. [61] The antibacterial effects of Ag salts have been noticed since antiquity,[62] and Ag is currently used to control bacterial growth in a variety of applications, including dental work, catheters, and burn wounds.[63, 64] In fact, it is well known that Ag ions and Ag-based compounds are highly toxic to microorganisms, showing strong biocidal effects on as many as 12 species of bacteria including E. coli.[65] Recently, Mecking and co-workers showed that hybrids of Ag nanoparticles with amphiphilic hyperbranched macromolecules exhibited effective antimicrobial surface coating agents.[66] Reducing the particle size of materials is an efficient and reliable tool for improving their biocompatibility. In fact, nanotechnology helps in overcoming the limitations of size and can change the outlook of the world regarding science.[67] Over the past few decades, nanoparticles of noble metals such as silver exhibited significantly distinct physical, chemical and biological properties from their bulk counterparts. Nano-size particles of less than 100 nm in diameter are currently attracting increasing attention for the wide range of new applications in different fields of industry. Such powders can demonstrate properties that differ substantially from those of bulk materials, as a result of small particle dimension, high surface area, quantum confinement and other effects. Most of the unique properties of nanoparticles involve not only the particles to be of nano-sized, but also the particles be detached without agglomeration. Discoveries in the past decade have clearly demonstrated that the electromagnetic, optical and catalytic properties of silver nanoparticles are strongly influenced by shape, size and size distribution, which are often varied by varying the synthetic methods, reducing agents and stabilizers. Accordingly, this review presents different tools of preparation silver nanoparticles and application of these nanoparticles in unlike fields.[68] Nanotechnology is predictable to open some new aspects to fight and avoid diseases using atomic scale tailoring of materials. The ability to uncover the structure and function of biosystems at the nanoscale, stimulates research leading to improvement in biology, www.wjpps.com Vol 4, Issue 04, 2015. 661 Poonam World Journal of Pharmacy and Pharmaceutical Sciences biotechnology, medicine and healthcare. The size of nanomaterials is alike to that of most biological molecules and structures; therefore, nanomaterials can be helpful for both in vivo and in vitro biomedical research and applications. The assimilation of nanomaterials with biology has led to the development of diagnostic devices, contrast agents, analytical tools, physical therapy applications, and drug delivery vehicles. In all the nanomaterials with antibacterial properties, metallic nanoparticles are the most excellent. Nanoparticles increase chemical activity due to crystallographic surface structure with their large surface to volume ratio. The significance of bactericidal nanomaterials study is because of the increase in new resistant strains of bacteria against most potent antibiotics. This has promoted research in the well known activity of silver ions and silver-based compounds, together with silver nanoparticles. This effect was size and dose dependent and was more well-defined against gram-negative bacteria than gram-positive organisms.[69] A major problem in medicine is the large number of infections associated with implanted and indwelling devices. Silver coating of medical devices is believed to preserve infection resistance. Several in vitro and animal studies as well as clinical observations on silver-nylon, silver-intra medullary pins, silver-oxide-Foley catheters and silver-coated vascular protheses have been interpreted as successful for the prophylaxis of foreign-body infections. Nevertheless, these products have not been established in clinical use. In this study authors have been able to present physico-chemical and pharmacological data as well as simple microbiological experiments explaining the reduced anti-microbial activity of silver-ions in some biological fluids.[70] Different approaches have been used for preventing biofilm related diseases in health care settings. Many of these methods have their individual demerits, which include chemical based complications, emergent antibiotic resistant strains, etc. The development of biofilm is the hallmark feature of Staphylococcus aureus and S. epidermidis infection, which consists of multiple layers of bacteria encased within an exopolysachharide glycocalyx. Nanotechnology may provide the answer to penetrate such biofilms and reduce biofilm formation. Therefore, the aim of present study was to exhibit the biofilm formation by methicillin resistance S. aureus (MRSA) and methicillin resistance S. epidermidis (MRSE) separated from wounds by direct revelation applying tissue culture plate, tube and Congo Red Agar methods.[71] There is enlarged demand for superior disinfection methods due to microorganisms resistant to multiple antimicrobial agents. Several types of disinfectants are obtainable with different www.wjpps.com Vol 4, Issue 04, 2015. 662 Poonam World Journal of Pharmacy and Pharmaceutical Sciences properties but the suitable disinfectant must be with awareness selected for any specific application to get the desired antimicrobial effect. Antimicrobial effect of a commercial nanosilver product, NanoCid, against some foodborne pathogens was evaluated. Minimum inhibitory concentrations (MIC) were observed by monitoring the growth of bacteria at 600 nm, after 24 hours incubation at 35°C. Minimum bactericidal concentrations (MBC) were determined based on 3 log decrease in the feasible population of the pathogens after incubation of nutrient agar plates at 35°C for 24 hours. The required exposure time for 3 log reduction in the viable population of the tested pathogens was determined as the minimum exposure time for efficient bactericidal activity. The MIC values of Ag NPs against tested pathogens were in the range of 3.12-6.25 μg/mL. While Listeria monocytogenes showed the MIC value 6.25μg/mL, Escherichia coli O157:H7, Salmonella typhimurium and Vibrio parahaemolyticus all showed the MIC values of 3.12 μg/ mL. However, all the pathogens showed the same MBC value of 6.25μg/mL. To obtain an efficient bactericidal activity against E. coli O157:H7 and S. typhimurium, the exposure time should be at least ca. 6 hours., while this time was ca. 5 hours for V. parahaemolyticus and ca. 7 hours for L. monocytogenes. Silver nanoparticles showed great antibacterial efficacy on four important food borne pathogens. Therefore, Ag NPs could be a good alternative for clean-up and disinfection of equipment and surfaces in food-related environments.[72] In recent years the outbreak of re-emerging and emerging infectious diseases has been an important burden on global economies and public health. The growth of population and urbanization along with poor water supply and environmental hygiene are the main reasons for enhance in occurrence of contagious pathogens. Transmission of infectious pathogens to the community has caused outbreaks of diseases such as influenza, diarrhea (Escherichia coli), cholera (Vibrio cholera), etc throughout the world. The comprehensive treatments of environments containing infectious pathogens using complex disinfectant nanomaterials have been proposed for prevention of the outbreaks. Among these nanomaterials, silver nanoparticles (Ag-NPs) with unique properties of high antimicrobial activity have attracted much interest from scientists and technologists to develop nanosilver-based disinfectant products. This article aims to review the synthesis routes and antimicrobial effects of Ag-NPs against various pathogens including bacteria, fungi and virus. Toxicology considerations of Ag-NPs to humans and ecology are discussed. Some current applications of Ag-NPs in water, air- and surface- disinfection are described. Finally, future prospects of Ag-NPs for treatment and prevention of currently emerging infections are discussed.[73] www.wjpps.com Vol 4, Issue 04, 2015. 663 Poonam World Journal of Pharmacy and Pharmaceutical Sciences In recent years, skin and soft-tissue infections (SSTIs), mainly due to multidrug-resistant pathogens are increasingly being encountered in clinical settings. Due to the development of antibiotic resistance and the outbreak of infectious diseases caused by resistant pathogenic bacteria, the pharmaceutical companies and the researchers are now searching for new unconventional antibacterial agents. Recently, in this field nanotechnology represents a modern and innovative approach to develop new formulations based on metallic nanoparticles with antimicrobial properties. The bacterial growth curve, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of silver nanoparticles (Ag-NPs) towards Staphylococcus aureus ATCC25923, methicillin-sensitive S. aureus (MSSA), and methicillin-resistant S. aureus (MRSA) were examined in this study. The experiment results showed that the lowest MIC and MBC of Ag-NPs to MRSA was 12.5μg/ml and 25μg/ml respectively. The obtained results suggested that Ag-NPs exhibit excellent bacteriostatic and bactericidal effect towards all clinical isolates tested regardless of their drug-resistant mechanisms.[74] A systematic and detailed study for size-specific antibacterial efficacy of silver nanoparticles (AgNPs) prepared using a co-reduction approach is showed here. Nucleation and growth kinetics during the synthesis process was precisely controlled and AgNPs of average size 5, 7, 10, 15, 20, 30, 50, 63, 85, and 100 nm were synthesized with excellent give up and monodispersity. Authors originate the bacteriostatic/bactericidal effect of AgNPs to be size and dose-dependent as determined by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of silver nanoparticles against four bacterial strains. Out of the tested strains, Escherichia coli and Staphylococcus aureus were found to be the mainly and least sensitive strains despite of AgNP size. For AgNPs with less than 10 nm size, the antibacterial efficacy was appreciably improved as revealed through delayed bacterial growth kinetics, corresponding MIC/MBC values and disk diffusion tests. AgNPs of the smallest size, i.e., 5 nm demonstrated the best results and mediated the fastest bactericidal activity against all the tested strains compared to AgNPs having 7 nm and 10 nm sizes at similar bacterial concentrations. TEM analysis of AgNP treated bacterial cells showed the presence of AgNPs on the cell membrane, and AgNPs internalized within the cells.[75] The antibacterial actions of silver nanoparticles (Ag-NPs) were considered by respect to Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli by observing the bacterial cells treated or not with Ag-NPs by field emission scanning electron microscope www.wjpps.com Vol 4, Issue 04, 2015. 664 Poonam World Journal of Pharmacy and Pharmaceutical Sciences (FE-SEM) as well as measuring the growth curves, development of bactericidal reactive oxygen species (ROS), protein leakage, and lactate dehydrogenase action concerned in the respiratory chain. Bacterial cells were treated with Ag-NPs powder, and the growth rates were investigated under varying concentrations of Ag-NPs, incubation times, incubation temperatures, and pH. As a result, S. aureus and E. coli were shown to be substantially inhibited by Ag-NPs, and the antibacterial activity of Ag-NPs did not fluctuate by way of temperature or pH. These outcomes recommend that Ag-NPs could be used as a valuable antibacterial material.[76] In this work authors observed the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a (111) lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag+ (in the form of AgNO3). Authors is projected that nanoscale size and the presence of a (111) plane combine to promote this biocidal property. To author’s knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and authors results reveal that silver nanoparticles undergo a shape-dependent relations with the gram-negative organism E. coli.[77] Authors have clarified the properties required for polymers that resist bacterial colonization for use in medical devices. The increase in antibiotic-resistant microorganisms has encouraged notice in the use of silver as an antimicrobial agent. Silver-based polymers can save from harm the inner and outer surfaces of devices in opposition to the attachment of microorganisms. Thus, this review focuses on the mechanisms of various silver forms as antimicrobial agents against different microorganisms and biofilms as well as the dissociation of silver ions and the resulting reduction in antimicrobial efficacy for medical devices. This author’s work suggests that the characteristics of released silver ions depend on the nature of the silver antimicrobial used and the polymer matrix. In addition, the elementary silver, silver zeolite and silver nanoparticles, used in polymers or as coatings could be used as antimicrobial biomaterials for a variety of hopeful applications.[78] Silver nanoparticles (AgNPs) with different shapes and sizes were chemically prepared and characterized by transmission electron microscope (TEM), UV-vis spectra and Fourier www.wjpps.com Vol 4, Issue 04, 2015. 665 Poonam World Journal of Pharmacy and Pharmaceutical Sciences transmission IR (FTIR). Their antibacterial activities against gram positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis) and gram negative bacteria (E. coli) were studied by authors. Antimicrobial activities of Ag nanoparticles had been increased with their larger surface area to volume ratio. Best antibacterial activity was experimental on using AgNPs prepared from sodium hypoborite (NABH4) as reducing agent and polyvinyl pyrrolidone (PVP) as stabilizer or protecting agent. It show zone of inhibition about 25 mm against S. aureus, 19 mm against S. epidermidis while the Cefoperazone antibiotic had inhibition zone of 15 mm. The same silver nano particles showed inhibition zone against E. coli of about 15 mm while the antibiotic had 12 mm inhibition zone. TEM revealed small size of silver nanoparticles (ranging from 1.5-3 nm) which stimulate biofilm construction and combined within this biofilm. They bind closely to the surface of microorganisms causing visible damage to the cells, and demonstrating good self-assembling ability.[79] ANTIMICROBIAL EFFECTS OF Se-NPs Selenium is an essential trace element in living organisms as integral part of seleno-enzymes. However, overload quantity of selenium is toxic for plants, animals and humans. The toxicity for plants depends on the capacity of synthesis of non-protein amino acids and also their volatilization in the form of dimethylselenide, while in animals on the rate of methylation and its excretion. In vitro studies showed that there are selenium-resistant animal and human celllines which showed altered selenium uptake. Exact mechanism of selenium toxicity remains unclear but there are many data about its prooxidant effect particularly in the form of selenite, while selenomethionine and selenocysteine are less toxic. Inorganic forms of selenium reacts with tissue thiols, such as glutathione to form selenotrisulphides and those are reacting with further thiols to generate oxygen free radicals, example superoxide anion. Organic diselenides are converted into selenols in presence of thiols which also results oxygen free radical generation. Another free radical hypothesis of selenium toxicosis is based on the methyl-selenide formation, which also outcome superoxide radicals and induce oxidative stress. Besides free radical formation selenium can have inhibitory property on thiol proteins, for instance those which have antioxidant affect.[80] The aim of this study was to evaluate by authors whether coating titanium discs with selenium in the form of sodium selenite decreased bacterial adhesion of Staphylococcus aureus and Staphylococcus epidermidis and impeded osteoblastic cell growth. In order to estimate bacterial adhesion, sterile titanium discs were coated with rising concentrations of www.wjpps.com Vol 4, Issue 04, 2015. 666 Poonam World Journal of Pharmacy and Pharmaceutical Sciences selenium and incubated with bacterial solutions of Staphylococcus aureus and Staphylococcus epidermidis and stained with Safranin-O. The effect of selenium on osteoblastic cell growth was also experiential. The adherence of MG-63 cells on the coated discs was detected by staining with Safranin-O. The quantity of covered area was calculated with imaging software. The tested Staphylococcus aureus strain exhibited a significantly reduced attachment on titanium discs with 0.5% and 0.2% selenium coating. Authors test strain from Staphylococcus epidermidis showed an extremely significant reduction in bacterial adherence on discs coated with 0.5% and 0.2% selenium solution. There was no inhibitory effect of the selenium coating on the osteoblastic cell growth. Selenium coating is a promising method to reduce bacterial attachment on prosthetic material.[81] The antibacterial property of lithium, selenium, and germanium were evaluated with high opinion to development, biofilm formation, and mutational frequencies (MF), of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. Selenium showed the greatest antimicrobial and bactericidal activities as exposed by zone of inhibition assay and scanning electron microscopy imaging (SEM). The SEM images showed that the metals in culture media led to cell disintegration that could have resulted in the leakage of cytoplasmic constituents, and cell dehydration. In biofilms, the combination of metal and antibiotics increased oxidative stress, mutational frequencies (MF), and formation of mutator phenotypes. Adding the antioxidant ascorbic acid reduced the S. aureus biofilm MF, but increased the MF of P. aeruginosa biofilm. Image analysis showed that metal and ascorbic acid cooperated in destroying cell structure pointing to the effectiveness of the antioxidant to prevent the formation of reactive oxygen species, oxidative stress, and bacterial DNA mutation rates. Usually, outcome showed that the antibacterial effect depended on the combinations of metal, antibiotic and antioxidant unlike and the bacterial strain being tested. Biofilm cultures yielded adherent colony variants that differed in appearance and in the capability of the variants to form hypermutator phenotypes with relatively high MFs. There was perfect sequence alignment of an approximately 500 bases of the P. aeruginosa of the wild-type and colony variants. In dissimilarity, the 16S rDNA sequence of S. aureus variant showed numerous mutations, deletions, and insertions, implying the high mutability of S. aureus when uncovered to external factors. Author’s studies should be conducted on the molecular basis of the antibacterial action and possible applications of selenium, germanium, and lithium in reducing antibiotic resistance, and biofilm infection. In calculation, there is a www.wjpps.com Vol 4, Issue 04, 2015. 667 Poonam World Journal of Pharmacy and Pharmaceutical Sciences need to know bacterial adaptation to metals and their communication with other antimicrobial agents in order to devise successful drug therapies.[82] Among the mainly complicated bacterial infections encountered in treating patients are wound infections, which may take place in burn victims, patients with traumatic wounds, necrotic lesions in people with diabetes, and patients with surgical wounds. Within a wound, infecting bacteria commonly build up biofilms. Many current wound dressings are impregnated with antimicrobial agents, such as silver or antibiotics. Diffusion of the agent(s) from the dressing may damage or destroy nearby healthy tissue as well as compromise the effectiveness of the covering. In dissimilarity, the antimicrobial agent selenium can be covalently friendly to the surfaces of a covering, prolonging its effectiveness. Authors examined the usefulness of an organoselenium coating on cellulose discs in inhibiting Pseudomonas aeruginosa and Staphylococcus aureus biofilm development. Colony biofilm process exposed that cellulose discs coated with organoselenium completely inhibited P. aeruginosa and S. aureus biofilm formation. Scanning electron microscopy of the cellulose discs established these results. Additionally, the coating on the cellulose discs was stable and effective after a week of incubation in phosphate buffered saline. These authors outcome demonstrate that 0.2% selenium in a coating on cellulose discs successfully inhibits bacterial attachment and biofilm formation and that, dissimilar other antimicrobial agents, longer periods of exposure to an aqueous environment don’t compromise the usefulness of the coating.[83] Antibiotic resistance in pathogenic bacteria is emerging as an issue of serious concern in biomedical research as well as food and health organizations. Metal complexes are currently being employed in medical devices for their inhibition to bacterial adherence and antibacterial activities. The primary plan of this study was to estimate the antibacterial activities of pure metals, including selenium, germanium and lithium on planktonic cultures and biofilms of three bacterial species: S. aureus, P. aeruginosa and E. coli. The antagonistic property of selenium, germanium and lithium on these three bacterial species were examined using zone of inhibition assay. The minimum inhibitory concentrations and minimum bactericidal concentrations of antibiotics (rifampicin, mupirocin and ciprofloxacin) and metals (selenium, germanium and lithium) were considered. The minimum biofilm eradication concentrations (MBEC) of metals were observed against biofilms composed of S. aureus and P. aeruginosa. Metal susceptibility tests suggested that biofilms displayed www.wjpps.com Vol 4, Issue 04, 2015. 668 Poonam World Journal of Pharmacy and Pharmaceutical Sciences increased resistance over their planktonic state. Differential inhibitory effects were observed for different strains of planktonic and biofilm bacteria in reaction to dissimilar metals and their changeable concentrations. Amongst the three metals tested, selenium proved to be the most active against all three species, whereas lithium demonstrated the least inhibitory effects. Scanning electron microscope (SEM) image analysis revealed numerous detrimental structural changes in bacterial cells exposed to metals compared to those developed in the metal-free culture. In conclusion, the outcome display the antibacterial efficacy of pure metals against planktonic and biofilm bacteria paving the way for additional alike investigations of alternative antibacterial agents. A recent study by Forceville and colleagues evaluated the effect of high-dose Se administration as a treatment for septic shock. The study was negative and conflicts with existing clinical data regarding Se administration in critically ill patients. Perhaps the key to understanding the differences linking these discrepant clarifications lies in considering the dose and timing of selenium administration.[84] Antimicrobial drug resistance remains an important difficulty in modern healthcare, impacting on action options, mortality, infection handle and financial issues. The beginning of new antimicrobial drugs has consistently been followed by the emergence of resistant bacteria. This review aims to response the problem of whether clinical enhancement is likely if action of Staphylococcus aureus infections is attempted with an antimicrobial drug against which resistance is expressed in vitro condition (RD). Over time, S. aureus has acquired a broad range of antimicrobial resistance mechanisms, and methicillin-resistant S. aureus (MRSA) strains have developed into the mainly ordinary multidrug-resistant healthcarerelated infection-causing bacteria in Europe. As intention-to-treat studies with an RD would be unethical, only observational studies to evaluate the impact of RD therapy have been performed. Most of these studies bolster the hypothesis that RD therapy offers no assistance to the patient, but some do not show a detrimental effect. Limited antimicrobial treatment options for rigorous, invasive infections caused by MRSA might tempt physicians to use antimicrobials to which in vitro resistance is reported by the microbiological laboratory. Reasons for this non evidence-based approach might include better pharmacokinetic/pharmacodynamic parameters, lower toxicity and better bioavailability in specific compartments, and/or the assumption of increased in vivo susceptibility of those microorganisms reported as resistant in vitro. In vitro resistance of a bacterium to a drug implies that exposing this bacterium to that drug should result in a worse clinical outcome than would be obtained with a drug to which resistance has not been experimental (SD). As a www.wjpps.com Vol 4, Issue 04, 2015. 669 Poonam World Journal of Pharmacy and Pharmaceutical Sciences counterpoint to in vitro resistance breakpoints, the concept of clinical breakpoints is therefore briefly revisited in this paper. In a nutshell, no evidence has been published that S. aureus infections can be reliably treated with RDs, either as a single organization or in combination therapy.[85] Staphylococcus aureus is a key bacterium commonly present in frequent infections. S. aureus infections are complex to treat due to their biofilm production and documented antibiotic resistance. Whereas selenium has been used for a extensive range of applications together with anticancer applications, the effects of selenium nanoparticles on microorganisms stay behind mostly unknown to date. The aim of this in vitro study was thus to observe the development of S. aureus in the occurrence of selenium nanoparticles. Results of this study provided the first evidence of strongly inhibited growth of S. aureus in the occurrence of selenium nanoparticles after 3, 4, and 5 hours at 7.8, 15.5, and 31 µg/mL. The percentage of live bacteria also decreased in the presence of selenium nanoparticles. Therefore, authors in this study suggests that selenium nanoparticles may be used to effectively prevent and treat S. aureus infections and thus should be further studied for such applications.[86] Sepsis is associated with an increase in reactive oxygen species and low endogenous antioxidative capacity. We postulated that high-conc. supplementation of sodium-selenite would recover the outcome of patients with severe sepsis and septic shock. 249 patients with severe systemic inflammatory response syndrome, sepsis, and septic shock and an Acute Physiology and Chronic Health Evaluation (APACHE) III score >70. Patients received 1000 µg of sodium-selenite as a 30-min bolus inoculation, followed by 14 daily constant infusions of 1000 µg intravenously. The most important end point was 28-day mortality; secondary end points were survival time and clinical course of APACHE III and logistic organ dysfunction system scores. In adding up, selenium levels in serum, whole blood, and urine as well as serum gluthation-peroxidase-3 activity were considered. From 249 patients included, 11 patients had to be disqualified. The intention-to-treat analysis of the remaining 238 patients revealed a mortality rate of 50.0% in the placebo group and 39.7% in the selenium-treated. A further 49 patients had to be excluded before the final analysis because of severe violations of the study protocol. In the lasting 92 patients of the study group, the 28-day mortality rate was considerably reduced to 42.4% compared with 56.7% in 97 patients of the placebo group. In predefined subgroup analyses, the mortality rate was significantly reduced in patients with septic shock with disseminated intravascular coagulation as well as in the most critically ill www.wjpps.com Vol 4, Issue 04, 2015. 670 Poonam World Journal of Pharmacy and Pharmaceutical Sciences patients with an APACHE III score >102 or in patients with more than three organ dysfunctions. Whole blood selenium conc. and glutathione peroxidase-3 movement were within the upper normal range during selenium treatment, whereas they remained considerably low in the placebo group. There were no side effects observed due to high-dose sodium-selenite action. The adjuvant action of patients with over dose sodium-selenite reduces mortality rate in patients with harsh sepsis or septic shock.[87] FUTURE PROSPECTS Importantly, some future prospects for use of Ag-NPs-based nanoparticles for treatments of contagious diseases were discussed. For silver and selenium based NPs to be used in the field of infectious diseases treatment, however, further investigation is needed to determine how to safely design, use, and dispose of products containing silver without creating a new risk to humans or the environment. A new class of nanosilver and nanoselenium containing disinfectant nanoproducts will be promising for advanced environmental treatments including air disinfection, water disinfection, surface disinfection and personal hygiene; this will help to prevent the further outbreak of diseases. CONCLUSIONS Ag-NPs are one of the mainly striking nanoparticles for commercialization prospects. They have been extensively used for antimicrobial, electronic and biomedical yield. In this review, we provide an inclusive appreciative of the Ag-NPs from synthesis methods, antimicrobial effects against biofilm forming S. aureus and possible toxicology considerations of Ag-NPs to both humans and environment. The importance is also located on the antiseptic capacity of Ag-NPs nanoparticles with respect to ecosystem. An overview of some current applications for use of Ag-NPs in antiseptic applications was given and discussed. Selenium is an essential dietary nutrient for most animals and humans, which is incorporated into twelve or more known proteins or enzymes as an amino acid, selenocysteine. Fruits and vegetables generally contain very low levels of selenium. Selenodiglutathione is the most potent selenium compound against cancer cells and readily arrests their growth as compared to selenite and any other selenium compound. At higher levels of dietary intake many selenium compounds can become toxic. One of the studies used a redox selenium compound covalently attached to bacterial phage and peptides to kill bacteria through the generation of superoxide radicals. www.wjpps.com Vol 4, Issue 04, 2015. 671 Poonam World Journal of Pharmacy and Pharmaceutical Sciences REFERENCES 1. Feynman R. 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