Evaluation of antibacterial properties of “Silver

nanoparticles” against Methicillin resistance

Staphylococcus aureus

By

Tehreem Fatima

Roll No. BSBTM-15-20

Session (2015-19)

BIOTECHNOLOGY DIVISION

INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY

BAHAUDDIN ZAKARIYA UNIVERSITY

MULTAN

2018

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 TABLE OF CONTENTS

Sr. No. Contents Page. No.

1 Introduction

2 Historical status of silver based antimicrobial agents

2.1 Synthesis of silver nanoparticles

2.2 Properties of silver nanoparticles

2.3 Applications of silver nanoparticles

3 Study objectives

4 Methods to achieve these objectives

5 Materials

6 Methods

7 Time line

8 Statistical analysis

9 Future prospects

10 References

LIST OF TABLES

Sr. No. Contents Page. No.

1 Effect of AgNPs on different bacterial species.

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1 Introduction:

Methicillin resistance Staphylococcus aureus abbreviated as MRSA belongs to family

Micrococcaceae, which is a class of gram positive type of bacteria. This bacteria either
present in isolated form or when exist in dividing cell, it is present in the form of pairs means
present un-separated. This bacterium mostly resides on the skin on humans and result in very
difficult to treat infections like severe chest problems, wound infections, infections of bones,
different types of bacterial infections and damage to heart by infecting its endothelial lining.
These infections when present in severe form, lead to the death of patient.

Before 1950, different methods were adopted for the treatment of this S. aureus
related infections which include the use of benzyl penicillin which is an antibiotic. Soon after
1950s, these strains attain resistance against these antibiotics. Benzyl penicillin is actually B-
lactamase. It is seen that these species resistant to these antibiotics produce an enzyme which
is known as B-lactamase that causes the inactivation of B-lactam by the process of
hydrolysis. Further investigations lead to the discovery of methicillin in 1959 for the
treatment of these infections. As far as methicillin was used widely as an antibiotic clinically,
these bacterial species also develop resistance against methicillin. But this time the resistance
is not due to the hydrolysis of B-lactam. But this time many binding proteins of penicillin
were involved for their inactivation. As the time passed away, these strains attain resistance
against many antibiotics. At that time nanotechnology evolved for the treatment of these
severe types of infections.(Stapleton, P.D. and Taylor, P.W., 2002)

Nanotechnology is basically a technique in which Nano size particles where used.

Here the meaning of prefix “Nano” is about one billionth. These nanoparticles may be of
iron, silver, gold, platinum etc. To treat the infection related to MRSA, different nanoparticles
were used like gold, copper uranium etc. But silver is most abundantly used for this purpose
as it is very effective against the treatment of wound infections and severe abdominal
infections. It is also widely used in other fields of biotechnology.

2 Historical Status of Silver Based Antimicrobial Agents

Silver metal exists naturally in earth’s crust. It has high conductivity and can be
changed into wires and sheets. It is relatively more hard than gold. From pre-historic times, it
has a wide range of applications in different fields of life like photography, utensils making,
coins making and also for ornamental purposes etc.(Rai, M. et al.,, 2012).

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 In the past silver is used for the treatment of wounds.(Moyer, C.A. et al.,, 1965). As it
is very reactive, it gets attach to the bacterial membranes and brings out its distortion and thus
eventually causes the death of bacteria. Different types of silver compounds possess the
antimicrobial and antibacterial property. Some of these include:

Silver nitrate (AgNO3): this solid compound came to be known by different names in
different regions of the world like “Lunar caustic” in English, “Pierre infernalis” in French. In
1700, AgNO3 is used to cure different diseases including of bones and perianal abscesses.
(Lansdown, A.B., 2002). Later on its drops for eye infections are used in 1881. Also this
compound is used for the treatment of gonorrheal diseases.

Silver sulfadiazine (AgSD): Like silver nitrate, AgSD shows disease resistance ability to
different strains of bacteria. Again its mode of actions is same to silver nitrate. It is very
effective against different bacterial species including E. coli, pseudomonas species and
species of Klebsiella.

Silver zeolite: This compound is composed of silicates of aluminum and alkaline earth
metals .Here ions of aluminum are replaced by silver ions. In different countries of the world
especially Japan, the ceramics and building stones have coatings of this complex compound
to protect them from bacterial attacks. (Koizumi, W. et al.,, 2003, Rai, M. et al.,, 2009).
Some scientists reported that there are two ways by which this compound kills the bacteria.
First method is direct killing its cells. Second method involves the generation of relative
oxygen species (ROS). These species includes hydroxide ions and hydrogen peroxide etc.
These species affect the respiratory system of bacterial species thus causing its death.
Matsumura et al. (2003)

Silver nanoparticles: Silver nanoparticles are very small in size usually in order of 100nm. It
possesses antibacterial property against different bacterial strains. It is prepared through
different methods including physical, chemical and biological methods. Also it exhibits
different properties like optical ones etc.

Table No.1 Effect of AgNPs on Different Bacterial Species.(Rai, M. et al.,,

2012)

Sr. # Different types of Silver Target species References

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 1 Silver ions(Ag+) S. aureus and E .coli (Yu, M.-F. et al.,,
2000)
RNA virus
(Butkus, R. et al.,,
2004)

2 AgNPs on cotton fabrics S. aureus (Durán, N. et al.,,

2007)

3 Silver zeolite Escherichia coli Matsumura et al.

(2003)

4 Silver Staphylococcus aureus and Shah Verdi et al.

nanoparticles(AgNPs) Escherichia coli (2007)

Gade et al. (2010)

5 Nanofibres impregnated Staphylococcus aureus and Jun et al. (2007)

AgNPs Escherichia coli

6 AgNPs coated medical Staphylococcus aureus and Ki‐Young (2011)

devices Streptococcus mutans

7 Silver nitrate(AgNO3) Periodontal pathogens Spacciapoli et al.

(2001)

2.1 Synthesis of Silver Nanoparticles

Nanoparticles are synthesized b using many different physical and chemical methods
which are very easy and economical. These methods are also very useful in maintaining and
restoring the reactive properties of nanoparticles. Likewise, silver nanoparticles are formed
by following processes:

2.1.1 Physical Method: Nanoparticles are being formed by physical methods which
includes the evaporation and condensation stages. These stages are carried out in a furnace
with normal atmospheric pressure. Many different types of nanoparticles are formed by this
physical method including silver, gold, platinum etc. but silver formed by this method has

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many disadvantages as this is a very time taking process. Further, it leads to the increase of
temperature. To overcome these drawbacks, another method is used for the synthesis of silver
nanoparticles which is laser ablation of metallic substances.

2.1.2 Chemical Method: The chemical method used for the synthesis of silver
nanoparticles is the reduction process. The reduction may be of organic and inorganic
substances. The substances involved are mainly the reducing reagents like ascorbate, Tollens
reagent, sodium citrate etc. In chemical process, silver ions are being reduced to metallic
silver by reducing agents.

2.1.3 Biological Method: Silver nanoparticles are also synthesized biologically. Here
different microbes are used. In one method, E. coli is used for the synthesis of silver
nanoparticles. E. coli is taken at room temperature. The liquid present on broth media is
inoculated for one night. After that about 1mM of silver nitrate is added to that liquid. After
about ten minutes, one can observe the formation of silver nanoparticles as the colour
changed to reddish brown. When held for a long time, the color changes from pale yellow to
light brown. From light brown to light reddish brown. And finally the colour changes to dark
reddish brown. So here we concluded from this color transition that more the time given,
more silver nanoparticles will be formed.

2.2 Properties of Silver Nanoparticles

Today, humans are affecting by a large number of bacterial diseases which may be
bacterial, viral or pathogenic infections. So to treat these infections, many types of metallic
nanoparticles are being used. They are important as they possess catalytic anti-bacterial
properties. Most importantly, silver nanoparticles are used for this purpose as it possesses
strong antimicrobial activity.

1) Silver nanoparticles also play a very vital role as far as their optical properties are
concerned. They undergo the phenomena of surface Plasmon resonance. Also it is
being used in different types of sensors like chemo sensors and biosensors.
2) In the field of biology, these nanoparticles are very useful against many bacterial
strains as they stick to their membranes and entered into body and cause the death of
bacteria. Silver nitrate was firstly used as antibacterial agent before the discovery of
silver nanoparticles.

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 3) When there is an implantation of joints, then silver nanoparticles are used for the
coating of that implant.(Abbasi, E. et al.,, 2016)

2.3 Applications of Silver Nanoparticles

Silver nanoparticles because of their distinctive properties, have wide range of

applications. They are being used as antimicrobial agents, low cost paper batteries, food
storage and many environmental applications. Some other used of silver nanoparticles
include:

1) They possess toxicity against many organisms.

2) They are used in cosmetics.
3) Silver nanoparticles are used to make detergents.
4) Also used to make sprays and other products which help to stop the widespread of
germs.
5) Being used in medical field for wound treatment.
6) They possess the catalytic activity.
7) The optical applications of silver involve the use of biosensors and also chemo-
sensors.

3 Study Objectives

The main and the foremost objective of this study are to:

1) Investigate the antimicrobial role of silver nanoparticles against methicillin resistance

Staphylococcus aureus (MRSA).
2) How silver nanoparticles are used for the treatment of wound infections.
3) What is the best concentration of silver nanoparticles that effectively inhibit the
growth of S. aureus?

4 Methods to Achieve These Objectives

From the time being immemorial, humans have being suffering from a number of
diseases. These diseases may be caused by viral strains or bacterial pathogens. Different
types of antibiotics are discovered to treat these infections like penicillin and methicillin.
With the advent of time, these bacterial strains developed resistance against these antibiotics.
Now at that time different types of metallic nanoparticles are used for the treatment of these

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infectious diseases like silver. The size of these particles ranges from 1 to 100mm apart.
Moreover, food items are also being conserves in containers that contain traces of silver
particles to avoid the bacterial growth as silver possess inhibitory effects against 15 bacterial
species. Silver nanoparticles have large surface area for bactericidal activity. Researches also
proved that silver is the most toxic element than others and possess the highest antibacterial
activity. The strength of silver is given as Co< Pb< Cr< Cd< Cu< Hg< Ag.

In case of wound infections, Staphylococcus aureus is the most tolerant bacteria as it

tolerates the harsh environment. This bacterium resides on many parts of the body like skin,
mouth and vagina. During the treatment of wounds, biofilms are used as they allow the
healing of wounds. But these bacteria are tolerant to that chemicals from which these
biofilms are made. So silver biofilms are used to inhibit the growth of bacteria over the
wounds and speed up the process of healing.

5 Materials

5.1- To check the antimicrobial activity of solver nanoparticles against S. aureus an

experiment is performed and major material required during the process includes the bacterial
strains, silver Nano composite film, stock solution, McFarland standard solution, 0.85% w/v
saline solution, Baird parker agar, Tryptic Soy broth, 0.11% sodium chloride, rotatory shaker
(250 rpm), sterile swabs, scanning electron microscope, gold plated nanoparticles.(Kim, S.-H.
et al.,, 2011)

5.2- Again to check the bactericidal effects of silver nanoparticles, another experiment is
performed and the materials used during that experiment includes the ultra-sonic cleaner,
transmission electron microscope (TEM), copper grid, carbon film, agar plates, Luria-Bertani
(LB) medium, high angle annular dark field detector (HAADF), PBS buffer, 250kv voltage,
glutaraldehyde vapours. (Morones, J.R. et al.,, 2005)

6 Methods:

6.1- Nanofilms are the thin membranes on which particular metal is coated. Silver nanofilms
contain silver in concentration of 1, 3, 5 and 7 ppm respectively. The major precaution here is
to protect these films from sunlight to sustain its antimicrobial activity. Further here strains of
S. aureus, a gram positive type of bacteria, are used to check the efficacy of silver Nano films
against it. For this reason bacterial cultures were held under -85оC which comprises a stock

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solution. Before sub culturing this bacterium on another plate, these bacteria are incubated at
40оC for two to three days. For getting the target colonies, these bacterial strains were also
sub-cultured on the same broth for 48 to 52 hours again at 40оC in the solution of saline. Here
bacterial inoculum was taken and sub culturing is performed. From this culture, plates were
taken and placed on agar medium. These petri plates containing inoculum and silver
nanofilms are incubated for one whole day at 40оC. Thus finally the antimicrobial tendency
of silver films were observed and determined by taking into account the inhibition area
around the films.

Further under this scheme another experiment is performed which is called as disk
diffusion method in which nanoparticles of silver were used for testing their antibacterial
ability against strains of bacteria. Before performing this experiment, antibacterial potency of
nanofilms of silver metal is checked by preparing fresh media of selected bacteria. Here the
material used for forming the inoculant is sodium chloride, McFarland and standard solution.
After this, gauze was prepared which had the definite size of 4cm each size. Then dip this
gauze into liquid media which contains the desired bacteria against which antibacterial
activity of silver nanoparticles was checked. Small pieces of gauze were dipped into media
and placed into petri dish. With the help of spatula, sub culturing is done on agar medium.
This step should be performed within 36 hours. After that, area of inhibition was observed
and registered.

6.2- In this experiment, nanoparticles of silver were used for the purpose of inhibition of
coalescence, present inside a carbon matrix. These nanoparticles were sling into water so that
there will be interaction between bacterial strains and silver nanoparticles. For the purpose of
homogenization, ultrasonic cleaner is used. After doing homogenization, few droplets of that
mixture was placed under the transmission electron microscope with the voltage of about
250kv.

In the first step, AgNPs of different concentrations like 0, 30, 60µg/ml was
investigated in comparison with each bacteria. After this, from different amount of AgNP,
different agar plates were prepared which also contains Luria-Bertani broth media. The
experimental conditions set up here are temperature 40оC and at 600nm optical density of 0.8.

The connection between bacteria and nanoparticles is analyzed at density of about

0.8nm in Luria bertani medium when the growth of bacteria is maximum. To form
homogenous mixture, AgNPs are added at temperature 40оС. After this the mixture is

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centrifuged at 6℃ for about five minutes with 3500 resolutions per minute. The cells that
have gone centrifugation are washed and added to PBS buffer. Following this procedure
about 8ul sample was put on a carbon film and then bring in contact with vapours of
glutaraldehyde for three hours. The main purpose of this step is the fixation of bacterial
samples. The analysis of AgNP’s position and structure of bacterial species after their
treatment with nanoparticles is done through JEOL 2010-F transmission electron microscope
at voltage of 25s0kv in the presence of high angel annular dark field detector.(Morones, J.R.
et al.,, 2005)

7 Time Line

During one year of research, following steps are to be performed to complete the
research project. These steps includes the problem identification, review of literature,
synopsis writing, performing experiments and waiting for their results, thesis writing,
analysis of the data gained through experimental work, writing the research paper and at the
end the viva of research paper. These steps are performed in couple of months starting from
June and ending in June of next year thus completing the one year of research.

CC

Problem Identification

Literature Review

R O
Synopsis
O
Experimental Results

Data Analysis

Research Paper

Thesis Writing

Thesis Viva

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8 Statistical Analysis

From statistical point of view, it is inferred that certain parameters of silver

nanoparticles are effective against bacterial species like their size, optical properties etc.
Small size provides high surface to volume ratio which because of its interaction with
bacterial species, is greatly enhanced. Morones and Raimondi also reported in 2005 that
bacterial activity of nanoparticles of silvers depends on its size and the size at which they
possess the greatest antimicrobial activity is 25nm. Also it is reported that triangular shaped
particles are more effective. (Pal, S. et al.,, 2007)Further studies revealed that concentration
of about 75µg/ml is also effective against the growth of bacteria.(Morones, J.R. et al.,, 2005)
Also they are being used as biosensors and chemo sensors in different fields of life. Infections
related to biofilms are also treated by AgNPs. Thus it is said that they are powerful weapon
against different bacterial species.

9 Future Prospects

There is a growing concern about the use of silver nanoparticles. It is widely used in
different fields of life. Most importantly, it is used in medical field where it has a wide range
of application. Especially it is used to treat very serious bacterial and fungal diseases which
would otherwise very difficult to treat. Further silver coated medical equipment are used so
that the spread of bacterial infections can be minimized to its greater extant. Silver
nanoparticles are also used to treat wound infections. Moreover, its dressings are also used to
avoid the contaminations from bacterial species. Also the biofilm related infections are
successfully cured by treating with silver nanoparticles.

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References

Abbasi, E., et al. (2016) Silver nanoparticles: synthesis methods, bio-applications and
properties. Critical reviews in microbiology. 42(2): 173-180.
Butkus, R., et al. (2004) Progress in chirped pulse optical parametric amplifiers. Applied
Physics B. 79(6): 693-700.
Durán, N., et al. (2007) Antibacterial effect of silver nanoparticles produced by fungal
process on textile fabrics and their effluent treatment. Journal of biomedical nanotechnology.
3(2): 203-208.
Kim, S.-H., et al. (2011) Antibacterial activity of silver-nanoparticles against Staphylococcus
aureus and Escherichia coli. Korean J. Microbiol. Biotechnol. 39(1): 77-85.
Koizumi, W., et al. (2003) Phase I/II study of S-1 combined with cisplatin in patients with
advanced gastric cancer. British journal of cancer. 89(12): 2207.
Lansdown, A. B. (2002) Silver I: its antibacterial properties and mechanism of action.
Journal of wound care. 11(4): 125-130.
Morones, J. R., et al. (2005) The bactericidal effect of silver nanoparticles. Nanotechnology.
16(10): 2346.
Moyer, C. A., et al. (1965) Treatment of large human burns with 0.5% silver nitrate solution.
Archives of surgery. 90(6): 812-867.
Pal, S., et al. (2007) Does the antibacterial activity of silver nanoparticles depend on the
shape of the nanoparticle? A study of the gram-negative bacterium Escherichia coli. Applied
and environmental microbiology. 73(6): 1712-1720.
Rai, M., et al. (2012) Silver nanoparticles: the powerful nanoweapon against multidrug‐
resistant bacteria. Journal of applied microbiology. 112(5): 841-852.
Rai, M., et al. (2009) Silver nanoparticles as a new generation of antimicrobials.
Biotechnology advances. 27(1): 76-83.
Stapleton, P. D. and Taylor, P. W. (2002) Methicillin resistance in Staphylococcus aureus:
mechanisms and modulation. Science progress. 85(1): 57-72.
Yu, M.-F., et al. (2000) Strength and breaking mechanism of multiwalled carbon nanotubes
under tensile load. Science. 287(5453): 637-640.

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