Comparative Study on Bactericidal Effect of Silver Nanoparticles, Synthesized Using Green Technology, in Combination with Antibiotics on Salmonella Typhi

doi:10.4236/jbnb.2012.34049

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Journal of Biomaterials and Nanobiotechnology, 2012, 3, 480-485

http://dx.doi.org/10.4236/jbnb.2012.34049 Published Online October 2012 (http://www.SciRP.org/journal/jbnb)

Comparative Study on Bactericidal Effect of Silver

Nanoparticles, Synthesized Using Green Technology, in

Combination with Antibiotics on Salmonella typhi

Shweta Rajawat, Mohammad Shums Qureshi

Department of Physics, Maulana Azad National Institute of Technology, Bhopal, India.

Email: [email protected], [email protected]

Received March 12th, 2012; revised June 25th, 2012; accepted July 24th, 2012

ABSTRACT

In this work bactericidal study of silver nanoparticles was taken up in combination with two standard antibiotics, am-

picillin and gentamycin, for Salmonella typhi. The antibacterial activities of antibiotics were increased in the presence

of AgNPs against test strains. The higher enhancing effect was observed for ampicillin in comparison to gentamicin

against test strains. Silver nanoparticles were synthesized elctrolytically using silver wire of 99% purity as anode and

carbon rod wrapped with LDPE as cathode. Silver nitrate [of Merck] of 0.01N is used as an electrolyte. Here tea extract

is added as capping and mild reducing agent. The polyphenols theaflavins and thearubigins, present in tea perform the

role of stabilizing or capping agents due to their bulky and steric nature. A brown coloured colloidal solution of silver

nanoparticles is obtained. The as-synthesized silver nanoparticles were characterized using XRD, TEM and UV-Vis

spectroscopy.

Keywords: Silver Nanoparticles; Salmonella typhi; Tea Extract; Ampicillin; Gentamicin

1. Introduction

Historically, silver has been extensively used for both

hygienic and healing purposes (Cheng and Schluesener,

2008). With time, the use of silver has reduced as an

anti-infection agent due to the advent of antibiotics and

other disinfectants and the poorly understood mecha-

nisms of their toxic effects. However, resistance of bac-

teria to bactericides and antibiotics has increased in re-

cent years. Some antimicrobial agents are extremely irri-

tant and toxic. Hence there is a need to find ways to for-

mulate safe and cost-effective Biocidal materials. Previ-

ous studies show that antimicrobial formulations of silver

in the form of nanoparticles could be used as effective

bactericidal materials. Highly reactive metal oxide nano-

particles exhibit excellent biocidal action against Gram-

positive and Gram-negative bacteria [1]. Thus, the syn-

theses, characterization, functionalization of nanosized

particles open the possibility of formulation of a new

generation of bactericidal materials.

Researchers have found that nanosilver and antibiotics

used together had an extremely strong effect against

gram positive and gram negative bacteria. Recently, due

to the limitations of antibiotics, the synergetic effect of

silver nanoparticles with antibiotics has been studied

combining silver nanoparticles with different antibiotics

against gram positive and gram negative bacteria.

A variety of preparation routes have been reported for

the synthesis of metallic nanoparticles [2,3] notable ex-

amples include, reverse micelles process [4,5], salt re-

duction [6], microwave dielectric heating reduction [7],

ultrasonic irradiation [8], radiolysis [9], Solvothermal

synthesis [10], electrochemical synthesis [11,12], etc.

Chemical methods for metal nanoparticle fabrication

usually involve toxic chemicals, which are usually ex-

pensive and potentially dangerous for the environment

[13]. Green synthesis of nanoparticles is an easy, effi-

cient and eco-friendly approach. Among several synthe-

sizing methods, biosynthetic methods employing either

biological microorganisms or plant extracts have emerg-

ed as a simple and viable alternative to chemical syn-

thetic procedures and physical methods. The synthesis of

metal nanoparticles using biological systems is an ex-

panding research area due to the potential ap plications in

nano medicines. Plant extracts play an important role in

remediation of toxic metals through reduction of the

metal ions. Silver nanoparticle are synthesized from

various parts of the herbal plants like bark of Cinnamon

(Sathishkumar et al., 2009), Neem leaves (Tripathi et al.,

2009), Citrus limon (Prathna et al., 2011), Tannic acid

(Sivaraman et al., 2009) and various plant leaves (Song

Comparative Study on Bactericidal Effect of Silver Nanoparticles, Synthesized Using Green Technology,

in Combination with Antibiotics on Salmonella typhi 481

and Kim, 2008).

We describe a synthesis approach which is simple and

“green” for the synthesis of metallic nanostructures of

noble metal i.e. silver (Ag). The bactericidal effect of

as-synthesized silver nanoparticles is then studied for

combination with gentamycin and ampicillin for Salmo-

nella typhi. Salmonella typhi is an obligate parasite that

has no known natural r eservoir outside of humans. It is a

multi-organ pathogen that inhabits the lymphatic tissues

of the small intestine, liver, spleen, and bloodstream of

infected humans. Infection of S. typhi leads to the de-

velopment of typhoid, or enteric fever. Gentamicin is

active against a wide range of human bacterial infections,

mostly Gram-negative bacteria including Pseudomonas,

Proteus, Serratia and the Gram-positive Staphylococcus.

Ampicillin is a beta-lactam antibiotic that has been used

extensively to treat bacterial infections since 1961. It is

relatively non-toxic. The toxicity of silver nanoparticles

synthesized using green technology is comparatively

reduced.

2. Materials and Method

The experimental setup consists of a beaker filled with

40 ml of electrolyte silver nitrate (0.02 N) of MERK. The

silver nitrate was further diluted with triple deionised

distilled water to obtain a solution of 0.01 N (mol/l)

strength. Two electrodes: silver wire (99% pure) as an-

ode and carbon rod wrapped by LDPE (Low Density

Poly Ethylene) material as cathode was used. LDPE ma-

terial is used to collect silver nanoparticles produced in

the synthesis process so that they can be easily extracted.

The distance between the two electrodes is 1 cm. The

diameter of the silver wire is 1.04 mm and the diameter

of the carbon rod used is 4 mm. The length of the carbon

rod as well as silver wire is 4.5 cm. 2 ml of tea extract is

added to the electrolyte as a capping agent. The whole

assembly is placed on magnetic stirrer which keeps the

solution in the beaker stirring continuously. The process

is carried at room temperature (30˚C) for 2 hours con-

tinuously. A Daniel cell of 1.1 volt and 2 ohm internal

resistance is used as current source. A potentiometer pot

along with a rheostat is used in the circuit to increase the

resistance and obtain current of different values in mam-

peres. Copper wires are used to connect the components

of the circuit. All the parameters were same for the sam-

ples synthesized, except the current through the circuit.

Figure 1 shows the set up. The beaker in the set-up is

covered with silver foil with holes for the electrodes.

3. Results and Discussions

The as-synthesised silver nanoparticles were obtained in

the form of colloidal solution of light brown colour as

shown in Figure 2. The as-synthesized silver nanoparti-

cles were characterized using XRD. Figure 3 shows the

graph obtained. The XRD results show Face-Centred-

Structure of pure silver nanoparticles oriented in planes

{111}, {200}, {220 }, and {311}. The peaks obtained for

as-synthesized silver nanoparticles are well in accor-

dance with JCPDS file No. 04-0783. The intensive dif-

fraction peak from the {111} lattice plane of face-centred

cubic silver indicates that the particles are made of pure

silver and that their basal plane, i.e., the top crystal plane

Figure 1. Experimental setup.

Figure 2. Synthesized colloidal solution of silver nanoparti-

cles.

Comparative Study on Bactericidal Effect of Silver Nanoparticles, Synthesized Using Green Technology,

in Combination with Antibiotics on Salmonella typhi

482

Figure 3. XRD graph of as-synthesized silver nanoparticles.

is the {111} plane. This 2θ value reflection angle con-

firms the presence of silver nanoparticles.

The morphology and the crystal structure of synthe-

sized silver nanoparticles were examined using HR-TEM.

HR-TEM unit used had a 120 kV class high-resolution

transmission electron microscope JEM-1400, Jeol, Japan.

The sample was placed on the carbon coated copper grid,

making a thin film of sample on the grid and extra sam-

ple was removed using the cone of a blotting paper and

kept in grid box sequentially. The TEM images show that

the particles are well dispersed and spherical in shape.

The particle size is between 2 nm to 23 nm. The average

size of the particle is 10 nm. The as-synthesized silver

nanoparticles are polydispersed nanoparticles. Such va-

riation in shape and size of nanoparticles synthesized by

biological systems is common [14]. The spherical and

oval shape of the particle, as visible in Figure 4, is due to

the fact that when a particle is formed, in its initial state,

it tries to acquire a shape that corresponds to minimum

potential energy. The spherical and oval shapes corre-

spond to the state of minimum potential energy. Other

factors that play role in determining the shape of silver

nanoparticles are the fast rate of reaction and low con-

centration of silver nitrate solution as slower rate of reac-

tion [15] and higher concentration of silver nitrate [16]

leads to anisotropic silver nanoparticles.

The UV-vis spectroscopy was carried by Uv-vis spec-

trometer of Systronics. It is a dual beam spectropho-

tometer. The base solution was triple de-ionised distilled

water to sample was added in small concentration. The

UV-visible spectrum shows the formation of silver

nanoparticles as the peak maxima 522 n m which is char-

acteristic to silver nanoparticles. The specific character-

istic peak, as shown in the Figure 5, for silver nanoparti-

cles is due to the surface Plasmon resonance. The nano-

particles which are smaller than the wavelength of light

can produce a coherent resonance waves at a particular

Figure 4. TEM image of silver nanoparticles.

Figure 5. UV-Visible graph of silver nanoparticles.

absorbance wavelength which is in the visible range for

silver nanoparticles.

Silver is known for its antimicrobial properties and h as

been used for years in the medical field for antimicrobial

applications and even has shown to prevent HIV binding

to host cells [17,18-21]. Additionally, silver has been

used in water and air filtration to eliminate microorga-

nisms [22-24].The antibacterial activity of nanoparticle

along with antibiotics ampicillin and gentamicin was

tested against S. typhi. Figure 6 shows the zone of inhi-

bition against S. typhi.The antibacterial activities of

antibiotics increase in the presence of silver nanoparticles

against gram positive and gram negative bacteria. The

size of metallic nanoparticles ensures that a significantly

large surface area of the particles is in contact with the

bacterial cells. Such a large contact surface is expected to

enhance the extent of bacterial elimination [25]. The

mechanism of the bactericidal effect of AgNPs is that

they may attach to the surface of the cell membrane dis-

turbing permeability and respiration functions of the cell

Comparative Study on Bactericidal Effect of Silver Nanoparticles, Synthesized Using Green Technology,

in Combination with Antibiotics on Salmonella typhi 483

Figure 6. Zone of inhibition with silver nanoparticles.

Table 1. 1 ml sample + ampicillin and gentamycin [10 mcg]

[kept for 15 mins] and zone readings taken.

Zone for

sample +

ampicillin

Zone for

sample +

gentamicin

SR. No. Organism

P1 P2MEAN P1 P2MEAN

1. Salmonella

typhi 14 2017 13 1715

Plate 1 = P1; Plate 2 = P2.

[26]. Smaller AgNPs having the large surface area avail-

able for interaction would give more bactericidal effect

than the larger AgNPs [26]. It is also possible that A gNP s

not only interact with the surface of membrane, but can

also penetrate inside the bacteria [27]. Zone of inh ibition

test was done for identification of degree of inh ibition by

silver nanoparticles in combination with gentamicin and

ampicillin antibiotics. 1 ml of as-synthesized colloidal

solution of silver nanoparticles was taken with 10 mcg of

ampicillin and with 10 mcg of gentamicin for 15 minutes

separately. Zone readings were taken for both. The com-

parative study given in Table 1 show that the zone size

for sample with ampicillin for Salmonella typhi is larger

than the zone size for sample with gentamicin. Thus

sample is more effective in combination with ampicillin

for S. typhi in comparison to gentamicin.

4. Conclusion

Production of silver nanoparticles can be achieved through

different methods. Chemical approaches are the most po-

pular methods for the production. However chemical me-

thods cannot avoid use of toxic chemicals in synthesis

methods. There is a growing need to develop environ-

mentally friendly processes of nanoparticles synthesis

that do not use toxic chemicals. So the approach to elc-

trolytically deposit highly pure silver nanoparticles with

the plant extract like tea (containing antioxidant compo-

nents) as capping agent in present synthesis me- thod is

well justified.

Due to the antibiotic resistance developed by the bac-

teria it is very hard to manage different types of antibac-

terial drugs. There is great need of agents to kill bacteria

and other microorganisms [28]. Silver nanoparticles have

been reported to have antimicrobial activity against a

wide range of microorganism. The use of silver nanopar-

ticles antimicrobial effects are highly sought after be-

cause of its broad spectrum activity, high rate of effect-

tiveness, and low cost. Research is being done to find

superior forms of silver-based antimicrobial agents.

The combination effect of nanosilver and ampicillin

has more potential compared to the other antibiotics and

may be caused by both, the cell wall lysis action of the

ampicillin and the DNA binding action of the silver

nanoparticles (Fayaz et al., 2009). The antibiotic mole-

cules contain many active groups such as hydroxyl and

amino groups, which reacts easily with silver nano-

particles by chelation, for this reason, the synergistic ef-

fect may be caused by the bonding reaction with anti-

biotic and silver nanoparticles.

Therefore a study for the combination of silver nano-

particles with most practised antibiotics i.e. ampicillin

and gentamicin was undertaken with S. typhi which shows

that the combination of silver nanoparticles and ampicil-

lin is more effective than th e combination of silver nano-

particles and gentamicin for Salmonella typhi.

5. Acknowledgements

Author is thankful to the Director, M.A.N.I.T., Bhopal,

India for the financial support in the form of scholarship

and the facilities at the institute, RRCAT Indore for XRD

facility, HSADL, Bhopal for the TEM facility, Micro Bio

laboratory Mumbai for bactericidal studies.

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