Antibacterial Characterization Studies of Silver...

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. .



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



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(



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.



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).



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).

.



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

.



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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