A review on synthesis and their antibacterial activity of silver and selenium nanoparticles against biofilm forming staphylococcus aureus

www.wjpps.com Vol 4, Issue 04, 2015. 652
Poonam World Journal of Pharmacy and Pharmaceutical Sciences
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
Antibacterial agents are very important in the textile industry, water
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
metal oxide nanoparticles (NPs) is increasing. This review focuses on
the Preparation and their potential with good antimicrobial activity of
Ag-NPs and Se-NPs against biofilm forming S. aureus. Such improved
antibacterial agents locally destroy bacteria, without being toxic to the
surrounding tissue. We also provide an overview of opportunities and
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
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 2.786
Volume 4, Issue 04, 652-677. Review Article ISSN 2278 – 4357
Article Received on
29 Jan 2015,
Revised on 22 Feb 2015,
Accepted on 18 March 2015
*Correspondence for
Author
Poonam Verma
School of Biotechnology,
IFTM University,
Moradabad, Uttar
Pradesh, India.

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

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, 42, 43, 44, 45]
and Burkholderia cepacia and
Chromobacterium violaceum in vivo.[46, 47]
The paradox of selenium (Se) is that it is both

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

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]

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 Fig 4: SEM image of Se-NPs

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 trouble-
free 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]

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

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 nano-
Ag 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

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,

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

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]

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

(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

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 cell-
lines 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

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

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 bio-
medical 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

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 healthcare-
related 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

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

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.

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