antibacterial characterization studies of silver...
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International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:16 No:06 1
162406-7575- IJBAS-IJENS @ December 2016 IJENS I J E N S
Antibacterial Characterization Studies of Silver Nanoparticles Against Staphylococcus Aureus and
Escherichia Coli
Sahar E.Abo-Neima *#, Sohair M. El-Kholy
**
*Physics Department, Faculty of Science, Damanhour University,Egypt, #E-mail: [email protected]
** Medical Biophysics Department, Medical Research Institute, Alexandria University, Egypt.
Abstract-- Multi-drug resistance is a growing problem in the
treatment of infectious diseases and the widespread use of broad-
spectrum antibiotics has produced antibiotic resistance for many
human bacterial pathogens. The resistance of bacteria towards
traditional antibiotics currently constitutes one of the most
important health care issues with impacts in practice.
Overcoming this issue can be achieved by using antibacterial
agents with serious negative multimode antibacterial action.
Silver nano-particles (AgNPs) are one of the well-known
antibacterial substances showing such multimode antibacterial
action. Therefore, AgNPs are suitable candidates for use in
combinations with traditional antibiotics in order to improve
their antibacterial action. Advances in nanotechnology have
opened new horizons in nanomedicine, allowing the synthesis of
nanoparticles that can be assembled into complex architectures.
The present study was performed to investigate the antibacterial
activities of AgNPs between gram negative Escherichia Coli
(E.Coli) and gram positive Staphylococcus aureus (S.aureus)
bacteria, AgNPs were synthesized by physical method. 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. This study was
performed by observing the bacterial cells treated or not with
AgNPs on bacterial conductivity, antibiotic susceptibility, and
morphological cellular structure by transmission electron
microscope (TEM). Results indicated that E.Coli and S.aureus
treated with AgNPs can inhibit bacterial growth and significant
increase to antibiotic susceptibility that inhibitors to protein, cell
wall and DNA. Results of dielectric relaxation and TEM indicated
morphological changes. The obtained results suggested that Ag-
NPs exhibit excellent bacteriostatic and bactericidal effect
towards all clinical isolates tested regardless of their drug-
resistant mechanisms. It will be concluded that Ag-NPs could be
used as an excellent effective antimicrobial material on
microorganisms. Moreover, a very low amount of silver is needed
for effective antibacterial action of the antibiotics, which
represents an important finding for potential medical applications
due to the negligible cytotoxic effect of AgNPs towards human
cells.
Index Term-- S.aureus , E.Coli , AgNPs, TEM, antibiotic
susceptibility, DNA, dielectric relaxation.
INTRODUCTION
Human beings are often infected by microorganisms
such as bacteria, molds, yeasts, and viruses present in their
living environments. Because of the emergence and increase
in the number of multiple antibiotic-resistant microorganisms
and the continuing emphasis on health-care costs, many
scientists have researched methods to develop new effective
antimicrobial agents that overcome the resistances of these
microorganisms and are also cost-effective. Such problems
and needs have led to resurgence in the use of silver-based
antiseptics that may be linked to a broad-spectrum activity and
considerably lower propensity to induce microbial resistance
compared with those of antibiotics [1,2,3,4]. In particular,
silver ions have long been known to exert strong inhibitory
and bactericidal effects as well as to possess a broad spectrum
of antimicrobial activities [5].
Silver ions cause the release of K+
ions from bacteria;
thus, the bacterial plasma or cytoplasmic membrane, which is
associated with many important enzymes and DNA, is an
important target site of silver ions [6,7,8,9].When bacterial
growth was inhibited, silver ions were deposited into the
vacuole and cell walls as granules [10]. They inhibited cell
division and damaged the cell envelope and cellular contents
of the bacteria [11]. The sizes of the bacterial cells increased,
and the cytoplasmic membrane, cytoplasmic contents, and
outer cell layers exhibited structural abnormalities. In addition,
silver ions can interact with nucleic acids [12]; they
preferentially interact with the bases in the DNA rather than
with the phosphate groups, although the importance of this
mechanism in terms of their lethal action remains unclear [13,
14, 15].
The effects of Ag-NPs on bacteria cell are
complicate. However, direct morphological observation by
electro-microscope gives us structural change on the bacterial
cell. It may give us useful information for understanding
antibacterial activity of silver nanoparticles on gram positive
Staphylococcus aureus (S. aureus) and gram negative
Escherichia coli (E. coli) were widely used to bacterial
experiment .S. aureus and E. coli live on the body surface of
mammal and sometimes occur infection to them. Furthermore,
they show their unique cell envelope structure of gram
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positive and gram negative bacteria. Therefore, S. aureus and
E.coli strains were selected for this antibacterial study. In this
study, AgNPs were evaluated for their applicability in
increasing antibacterial activities against S. aureus and E. coli.
Nanotechnology nowadays is their ability to offer the
opportunity to fight microbial infections via synthesis of
nanoparticles. The mechanism of prevention of bacterial
growth by antibiotics is quite different from the mechanisms
by which nanoparticles inhibit microbial growth. Therefore,
nanoparticles have the potential to serve as an alternative to
antibiotics and to control microbial infections [16,17]. Recent
studies have demonstrated that specially formulated metal
oxide nanoparticles have good antibacterial activity and
antimicrobial formulations comprising nanoparticles could be
effective bactericidal materials [18, 19].
Synthesis of nanosized drug particles with tailored
physical and chemical properties is of great interest in the
development of new pharmaceutical products. Silver is a
nontoxic, safe inorganic antibacterial agent and is capable of
killing about 650 types of diseases causing microorganisms
and its ability to exert a bactericidal effect at minute
concentrations [20, 21]. Silver has the advantage of having broad antimicrobial activities against gram-negative and
gram-positive bacteria and there is also minimal development
of bacterial resistance [22, 23].Silver ions have long been
known to have strong inhibitory and bactericidal effects as
well as a broad spectrum of antimicrobial activities; Some
forms of silver have been demonstrated to be effective against
burns, severe chronic osteomvelitis, urinary tract
infections[23].
The antimicrobial activity of silver has been
recognized by clinicians for over 100 years [24]. In addition,
many reports showed that Hippocrates recognized the role of
silver in the prevention of disease and the romans stored
wine in silver vessels to prevent spoilage. However, only in
the last few decades the mode of action of silver as an
antimicrobial agent has been studied without any rigour
[25].
Silver has a significant potential for a wide range of biological applications such as antifungal agent, antibacterial agents for antibiotic resistant bacteria, preventing infections,
healing wounds and anti-inflammatory [18].Silver ions Ag+
and i t s compounds are highly toxic to microorganisms
exhibiting strong effects on many species of bacteria but have
a low toxicity towards animal cells. Therefore, silver ions, being as antibacterial component, silver ions inhibited cell
division and damaged the cell envelope and cellular contents
of the bacteria [26].The size of the bacterial cells increased the
cytoplasmic membrane, cytoplasmic contents and the outer
cell layers exhibit abnormalities. In addition ionic silver
strongly interacts with thiol groups of vital enzymes and with
the bases in the DNA and inactivates them so that DNA loses
its replication ability once the bacteria are treated with silver
ions [27, 28] and the bacterial cell death. The effects of Ag-
NPs on the morphological structural of bacterial cells were
observed by TEM.
The main objective of this work to study the
interaction of bacteria with silver nanoparticle to evaluate the
antibacterial activity of AgNPs against bacteria. It appears that
t h e c o m b i n e d toxic effect of silver and hydrogen peroxide
may be related with damage to cellular proteins. However, the
mechanism of antimicrobial effects o f silver is still not fully
understood. The effects of silver i o n s on bacteria may b e
c o mp l i c a t e d ; however direct observation of the
morphological a nd structural c h a n g e s may provide useful
information for understanding the comprehensive antibacterial
effects and t h e p r o cess of inhibition of silver ions.
MATERIALS AND METHODS
EXPERIMENTAL SYSTEM MATERIALS USED TO PREPARE COLLOIDAL SILVER
Silver wires (Gredmann ,99.99%, 1mm in diameter and
submerged in deionized water the DC arc-discharge system
consists of two silver electrodes 1mm in diameter, servo
control system which maintains a constant distance between
the electrodes, power supply system which controls the DC
arc-discharge parameters and Glass container with an
electrode holder and deionized water to collect the silver
colloids [29].
PREPARATION OF SILVER NANOPARTICLES
SUSPENSION IN PURE WATER
The power supply system provides a stable pulse
voltage for etching the silver electrodes in pure water. Silver
wires are used as both the positive and negative electrodes; the
pure silver wires are etched by the DC pulse arc-discharge in
pure water [30] .During silver nanoparticle production by arc
discharge in water, water decomposition (e.g. electrolysis) was
also observed. This results in generation of gaseous hydrogen
and oxygen, which appear in the water as small bubbles partly
dissolved in the water medium. Hydrogen and oxygen start to
interact with the newly prepared silver nanoparticles. Since
hydrogen (molecular or atomic forms) does not adsorb on
silver particle surfaces at room temperature [31], and also is
not significantly dissolved in water, it is ultimately removed
from the water suspension to the gas phase. An electricity
generator such as 13.5/30V AC power adapter 40 watt, two
insulated alligator clips to replace the plug on the end of the
power adapter. Two 12 cm lengths of 99.99 % of pure silver
strips (Fig.1a) and 100ml glass jar Fig.1b. .
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Fig. 1. (a) power supply (b) glass jar
CHARACTERIZATION OF Ag-NPs
The surface morphology and size of the AgNPs were
examined using a transmission electron microscope (TEM)
The particle size distribution and surface charge of AgNPs
were determined using particle size analyzer (Zetasizer nano
ZS, Malvern Instruments Ltd., U.K.) at 25°C with 90°
detection angle.
BACTERIAL STRAINS, REAGENTS AND CULTIVATION
The S.aureus (ATCC# 25923) and E.Coli (ATCC# 25922) cells used in the present study were supplied by
Ocology center in Damanhour, Egypt. Muller-Hinton broth
(Becton Dickinson, U.S.A) and Muller-Hinton agar (Becton
Dickinson, U.S.A) were used as a culture media. Muller-
Hinton broth contains beef extract powder, and acid digest of
casein, and soluble starch. Muller-Hinton agar contains agar in
addition to the above reagents mentioned in Muller-Hinton
broth composition [26, 32]. All other reagents used were of the
purest grade commercially available.
GROWTH CURVE OF BACTERIAL CELLS TREATED WITH
AG-NPS
To study the growth curves of bacterial cells exposed to Ag-NPs, Muller–Hinton broth with concentration
0.046gm/mole of Ag-NPs was used, and the bacterial cell
concentration was adjusted to 106CFU/ml [26]. Each culture
was incubated in a shaking incubator at 37oC for 24 h. Growth
curves of bacterial cell cultures were attained through repeated measures of the optical density (O.D) at 600nm using a spectrophotometer model (UV/visible spectrophotometer LKB-Nova spec, made in England), and the concentration of cells (number of cells CFU/ml) was determined by plate counting technique and appropriate dilutions of the bacterial cells were used to inoculate nutrient agar plates. Inoculated
plates were then incubated at 37°C for 24h by counting the
number of colonies developed after incubation and multiplying it with the dilution factor the number of cells in the initial population is determined with CFU/ml [17].
TRANSMISSION ELECTRON MICROSCOPY
The morphological changes of bacteria untreated and treated with Ag-NPs have been determined using Transmission Electron Microscope (TEM).TEM investigation
was done in TEM Unit, Faculty of science, Alexandria
University. Bacterial cells (10 -100µl) were fixed in 300µl of
glutaraldehyde (Merck, Darmstadt, Germany) and 3% 0.1 M
phosphate buffer saline (PBS) (Sigma,Steinheim, Germany),
pH= 6 in a microcentrifuge (Hettich Universal 30 RF, Hettich,
Tuttlingen,Germany), so that pellets no larger than 0.5 mm
thick were obtained. The supernatant fluid was decanted and
aspirated with a Pasteur pipette. The pellet was suspended in
same fixative and allowed to stand for 4 h. The fixative was
replaced with a phosphate b u f f e r s o l u t i o n ( 0.1M
P B S , pH=7.2). Centrifugation was followed by three washes
in phosphate buffer; each involved gentle suspension of the
bacterial cells with a Pasteur pipette and then prompts
sedimentation in the centrifuge. For postfixation the pellet was
then suspended in the microfuge tubes with 2% Os0 (Merck,
Darmstadt,Germany) in 0.1M PBS, pH 7.2, for 2h and then
progressively dehydrated with ethyl alcohol. After
dehydration, intact pellets were easily removed from the
microfuge tubes for embedding in Epon 812(FlukaChemie,
(Leica UltracutR,Vienna, Austria) stained with 2% uranyl
acetate (SPI_CHEM, West Chester, PA,USA) for 15 min at
60°C, then washed, and stained for 10 min at room
temperature with saturated lead citrate (FlukaChemie, Buchs,
Switzerland). The sections were examined by TEM [33].From
each sample 10 thin slices (approximately 100 nm) were cut
with a diamond knife and stained with uranyl acetate and lead
citrate on grids. Each of these sections was examined with a
Philips EM201 80 kV Transmission Electron Microscope and
images were taken with a 35mm camera [34, 35].
ANTIBIOTIC SUSCEPTIBILITY TEST (AST)
S.aureus and E.Coli bacterial cells were tested for their in vitro susceptibility to various antibiotics using the agar diffusion method. The antibiotics used in this study were chosen to represent different modes of action. These discs
were Amicacin [AK (30µg)], Ampicillin [AMP
(10µg)],Ceftriaxone [CRO (30µg) ],Cefuroxime [CXM
(30µg)], Ampicillin [AM (10µg)] Amoxicillin/Clavulanic acid
[AMC (30µg)], Ampicillin-Sulbactam [SAM(30µg)] and
Cefazolin [KZ (30µg)] which inhibitors for cell wall synthesis.
Also, Ciprofloxacin [CIP (5µg)], Ofloxacin [OFX (5µg)], and
Norfloxacin [NOR (10µg)] which are inhibitors for bacterial
DNA. In addition to [E (15µg)], Streptomycin [S(10µg)] and
Chloramphenicol [C (30µg)] which are inhibitors for the
proteins. After plate inoculation and incubation at 37oC for 24
h, the diameters of the inhibition or stimulation zone of treated
and untreated bacterial cells were measured in mm.
DIELECTRIC MEASUREMENTS FOR THE
BACTERIAL CELLS
For measurement a bacterial sample (1ml) was placed
into sterile micro centrifuge tube and centrifuged at 14,000
rpm at 4°C for 15 min. The pellet was then harvested and
suspended in a 1 mL volume of sterile deionized water. The
tube was then centrifuged and the pellet was washed with
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deionized water twice more, before finally being suspended in sterile deionized water. A fixed concentration of bacterial cells
(106CFU/mL) was used for all samples which were controlled
through the use of the spectrophotometer. The dielectric measurements were carried out for the samples in the frequency range 42Hz-5MHz using a loss Factor Meter type HIOKI 3532 LCR Hi TESTER; version 1.02, Japan, and cell
types (PW 950/60) manufactured by Philips. The cell has two parallel square platinum black electrodes of 0.8 cm side each,
and area 0.64cm2, with an inter-electrode distance of 1cm.
During the measurements both the cell and the sample were
kept at 25°C in an incubator (Kottermann type 2771,
Germany). Each run was repeated three times. The measured
values of capacitance, C, and resistance, R, were used to
calculate real (dielectric constant) from equation (1), and
imaginary parts (dielectric loss) of the complex permittivity
from equation (2) .The conductivity σ was calculated from the
equation(3)[36].
2f 0
STATISTICAL ANALYSIS
All experiments were repeated at least three times and
the statistical significance of each difference observed among
the mean values was determined by standard error analysis.
The results were represented as means ± SD. Data from
bacterial growth studies were compared using Student T-test
and ANOVA analysis, the level of significance was set at p <
0.05, which was considered statistically significant [37, 38].
RESULTS & DISCUSSION
Increasing hospital and community-acquired infections
due to bacterial multidrug-resistant (MDR) pathogens for
which current antibiotic therapies are not effective represent a
growing problem. Antimicrobial resistance is, thus, one of the
major threats to human health [39], since it determines an
increase of morbidity and mortality as a consequence of the
most common bacterial diseases [40]. Resistance genes have
recently emerged [41], favoured by improper use of antibiotics
[42]; hence, the first step in combating resistance envisions the
reduction of antibiotic consumption [43]. Antimicrobial
resistance is a complex mechanism whose etiology depends on
the individual, the bacterial strains and resistance mechanisms
that are developed [44]. The emergence of resistance against
newly developed antibiotics [45], further supports the need for
innovation, monitoring of antibiotic consumption, prevention,
diagnosis and rapid reduction in the misuse of these drugs. It is
thus necessary to optimize antibiotics’ pharmacokinetics and
pharmacodynamics in order to improve treatment outcomes
and reduce the toxicity and the risk of developing resistance
[46]. To address the problem of resistance, it will be necessary
to change the protocols of use of antimicrobials so that these
drugs are administered only when all other treatment options
have failed [42]; and joint efforts of governments and
academic networks are needed to fight against the globally
spreading of multidrug resistant pathogens. Today, there is a
need to seek alternative treatments [47]. Non-traditional
antibacterial agents are thus of great interest to overcome
resistance that develops from several pathogenic
microorganisms against most of the commonly used
antibiotics [42].
The extensive use of engineered nanoparticles with
antimicrobial properties and their increased release into the
environment have raised major concerns due to potential (eco)
toxicological effects and inappropriate testing methods. At
present, there are virtually no applicable standard
methodologies for evaluating the effects of exposure of
microbial biota to nanoparticles, regardless of whether these
are favourable effects (e.g. against pathogens) or adverse
impacts in the environment [48].
Silver Nanoparticles and Antibacterial Activity Nanoparticles are now considered a viable alternative to
antibiotics and seem to have a high potential to solve the
problem of the emergence of bacterial multidrug resistance
[49]. In particular, AgNPs have attracted much attention in the
scientific field [50,51]. Silver has always been used against
various diseases; in the past it found use as an antiseptic and
antimicrobial against gram-positive and gram-negative
bacteria [52, 53] due to its low cytotoxicity [54]. AgNPs were
considered, in recent years, particularly attractive for the
production of a new class of antimicrobials [42,55,56] opening
up a completely new way to combat a wide range of bacterial
pathogens. Although the highly antibacterial effect of AgNPs
has been widely described, their mechanism of action is yet to
be fully elucidated. In fact, the potent antibacterial and broad-
spectrum activity against morphologically and metabolically
different microorganisms seems to be correlated with a
multifaceted mechanism by which nanoparticles interact with
microbes. Moreover, their particular structure and the different
modes of establishing an interaction with bacterial surfaces
may offer a unique and under probed antibacterial mechanism
to exploit. From a structural point of view, AgNPs have at
least one dimension in the range from 1 to 100 nm and more
importantly, as particle size decreases, the surface area-to-
volume ratio greatly increases. As a consequence, the physical,
chemical and biological properties are markedly different from
those of the bulk material of origin.
TEM Analysis for Silver Nano-particles
Fig.2. shows the TEM image of the colloidal silver
nanoparticles, the size were investigated by TEM which
indicates that silver particles are ranged from 26-40 nm
fRC
2
A
Cd
0 )1( )2( )3(
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B
Ab
sorb
ance
at
60
0n
m
Bac
teri
al c
ou
nt
(CFU
/mL)
average diameter and spherical shape in a magnification×75k.
Fig.3. shows the image of the Colloidal silver nanoparticles of
total magnification x 150 k. .
Fig. 2. Colloidal silver Nanoparticles identified by (TEM) magnification x 75k.
Fig. 3. Colloidal Silver nanoparticles identified by TEM (magnification x 150 k).
Growth curves of bacterial cells treated with Ag-NPs The growth curves of bacterial cells treated with Ag-
NPs indicated that Ag-NPs could inhibit the growth. The
growth curve of Ag-NPs treated S.aureus cells are shown in
Fig 4(A&B). The growth curve of E.Coli cells treated with
Ag-NPs is shown in Fig 5(A&B). The results indicate that the
antibacterial activity of Ag-NPs could inhibit the bacterial
growth. The bacterial cell colonies on agar-plates were
detected by viable cell counts. Viable cell counts are the
counted number of colonies that are developed after a sample
has been diluted and spread over the surface of a nutrient
medium solidified with agar and contained in a petri dish. The
number of CFU reduced significantly with by using silver
nanoparticles. The bacterial growth inhibition trend observed
from CFU data has matched well with the results of optical
density.
A S.aureus without AgNPs
treated S.aureus with AgNPs
1
0.8
0.6
0.4
0.2
0
count S.aureus without AgNPs x109
count treated S.aureus with AgNPs x102
10
8
6
4
2
0 10 20 30
Incubation time (hour)
0 0 10 20 30
Incubation time (hour)
Fig. 4. (A&B) Growth curves of S.aureus cells exposed to AgNPs at normal condition as compared with control cells.
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Fig. 5. (A&B) Growth curves of E.Coli cells exposed to AgNPs at normal condition as compared with control cells.
increases the passive permeability and manifest itself as aEffects of Ag-NPs on morphology of bacterial cells
The morphological changes of bacterial cells were
observed by TEM. (Fig.6a) revealed the TEM of untreated
E.Coli which shows normal structure of cell membrane but
E.Coli treated with Ag-NPs Fig.(6.b,c,d) showed an
accumulations of membranous structure in the cytoplasm of
the biocides treated cells, and there is morphological
alternations were seen in the cells. The cytoplasm shows
granularity with regions where DNA fibrils are evident. Also
coagulate material were seen inside the treated cells.Fig.7.
revealed the TEM micrograph of untreated S.aureus which
shows a dark area where the sample had a high electron
density. The morphological investigation of the samples
treated with Ag-NPs were greatly different to those of the
untreated cells revealed a disintegration of the cell wall, Fig
(7b), the light area show where the sample had a low electron
density, extrusion of the cytoplasmic contents. Fig.(7.c,d,e)
revealed disruptions with release of intracellular material and
losing their cytoplasm and cell ghost is an empty intact cell
envelope structure devoid of cytoplasmic content including
genetic material. The distortion of the physical structure of the
cell could cause the expansion and destabilization of the
membrane and increase membrane fluidity, which in turn
leakage of various vital intracellular constituents, such as ions,
ATP, nucleic acids, sugars, enzymes and amino acids. In
Fig.(7.f ,g ,h) cell wall perforation with release of significant
intracellular components, projections of the cell wall and
release of membrane vesicles were observed. TEM
micrograph shows silver nanoparticles not only adhered at the
surface of cell membrane, but also penetrated inside the
bacterial cells, these reveals that nanoparticles have penetrated
inside the bacterial cells. Nanoparticles have larger surface
area available for interactions, which enhances bactericidal effect than the large sized particles; hence they impart
cytotoxicity to the microorganisms. The mechanism by which the nanoparticles are able to penetrate the bacteria is not
understood completely, but studies suggest that when E. coli
was treated with silver, changes took place in its membrane
morphology that produced a significant increase in its
permeability affecting proper transport through the plasma
membrane, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane, resulting
into cell death. It is observed that silver nanoparticles have
penetrated inside the bacteria and have caused damage by
interacting DNA. Silver tends to have a high affinity to react
with different compounds [27].
Fig. 6. TEM micrographs of untreated E.Coli (a) and E.Coli treated with Ag-NPs (b,c,d )(magnification × 20000).
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AK 18±0.53 27±0.33
SAM 6±0.26 11±0.12
KZ 6±0.45 11±0.42
CFR 9 ±0.63 12±0.52
Protein inhibitors
C 22±0.14 28±0.72
E 23±0.11 29±0.44
S 11±0.35 19±0.54
DNA inhibitors
NOR 27±0.32 33±0.12
CIP 21±0.6 31±0.18
OFX 23±0.45 28±0.12
Antibiotics Before treated treated
Cell wall inhibitors
AMP 10±0.41 14±0.25
CRO 7±0.64 10±0.16
CXM 7±0.76 8±0.14
AMC 9±0.62 14±0.12
AK 11±0.13 16±0.22
SAM 8±0.16 20 ±0.23
KZ 8±0..25 10 ±0.54
CFR 8 ±0.33 25±0.56
Protein inhibitors
C 9 ±0. 22 16 ±0.36
E 13±0.14 20±0.25
S 8 ± 0.32 22 ± 0.84
DNA inhibitors
NOR 7±0.42 8±0.12
CIP 11±0.7 30±0.18
OFX 8±0.15 15±0.12
Antibiotics Before treated treated
Cell wall inhibitors
AMP 11±0.31 13±0.22
CRO 7±0.14 18±0.12
CXM 7±0.16 11±0.11
AMC 9±0.32 13±0.32
Fig. 7. TEM micrographs of untreated S.aureus (a) and S.aureus treated with Ag-NPs (b,c,d,e,f,g and h) magnification × 20000).
Antimicrobial activity of Silver Nanoparticles Results o b t a i n e d by using d i s k diffusion tests w e r e
also good where AgNPs showed a clear zone of inhibition on agar plates spread with S . a u r e u s a n d E . c o l i s t r a i n s . Table.I& Table.II revealed the antibiotic sensitivity test results of S.aureus and E.coli are given for control samples and those treated with AgNPs. In this test 14 different antibiotics having different biological actions on the microorganism were used. It is clear from the Table.I& Table.II that the sensitivity of the two microorganisms treated
with AgNPs has been increased for all used antibiotics which
are inhibitors to cell wall, protein and DNA. Fig (8&9) shows
Inhibition zones for S.aureus and E.Coli unexposed and
treated with AgNPs respectively. The isolates showed
sensitivity to silver nanoparticles, these particles are dose
dependent and showed a higher activity levels. There wasstatistically significant effect of the nanoparticles remembered
at the p< 0.05 level.
S.aureus was more susceptible to AgNPs than E.coli
bacteria used in this work, while the highest effects at the used
silver concentration were observable for antibiotics
(AK,CRO,CXM,CRO,NOR,CIP,S,E and C) and
(CFR,SAM,AMC,AK,C,E,S,CIP and OFX) for S.aureus and
E.coli respectively. Moreover, a very low amount of silver is
needed for effective antibacterial action of the antibiotics,
which represents an important finding for potential medical
applications due to the negligible cytotoxic effect of AgNPs
towards human cells.
Table I
Antibiotic sensitivity of S. aureus before and after treated with Ag-NPs (Mean inhibition zone diameter in mm).
Table II Antibiotic sensitivity of E.Coli before and after treated with Ag-NPs
(Mean inhibition zone diameter in mm).
.
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Fig. 8. Histohraph for inhibitory zone diameter for unexposed S.aureus and treated with Ag-NPs.
Fig. 9. Histohraph for inhibitory zone diameter for unexposed E.Coli and treated with Ag-NPs
Dielectric relaxation properties Fig (10,11) and (12,13) illustrate the variation of
dielectric constant and dielectric loss plotted in one scale (left Y-axis) and conductivity S plotted in the right Y- axis, as a function of frequency, for both control bacterial samples and the treated with Ag-NPs. The figures show that the dielectric curves for both samples (untrated and treated with Ag-NPs) pass through dispersion in the frequency range 42Hz
to 5MHz.Moreover, the decrease of the values of as a function of the applied frequency is accompanied by increased in the values of electrical conductivity; this yields a
consistency test for the data as stated by Kramers-Krong
relations. The results also indicate pronounced difference in
the values of the dielectric properties of the treated samples as
compared with the control one. .
Fig. 10. Dielectric behavior of untreated. S.aureus
.
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Fig. 11. Dielectric behavior of S. aureus treated with Ag-NPs.
Fig. 12. Dielectric behavior of untreated. E.Coli
CONCLUSION
Fig.13. Dielectric behavior of E.Coli treated with Ag-NPs
The results of this work can be summarized into the
following: 1-It is believed that silver nanoparticles after penetration
into the bacteria have inactivated their enzymes, generating
hydrogen peroxide and caused bacterial cell death.
2- Silver nanoparticles have an excellent effect and
potential in reducing bacterial growth for practical applications.
3- Silver nanoparticles adhered to the cell wall of
bacteria and penetrated through the cell membrane. This
resulted into inhibition of bacterial cell growth and
multiplication.
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