International Journal of Veterinary Science and Medicine xxx (2017) xxx–xxx

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International Journal of Veterinary Science and Medicine

journal homepage: www.elsevier.com/locate/ijvsm
www.vet.cu.edu.eg

Full Length Article

Antibacterial effect of gold nanoparticles against Corynebacterium

pseudotuberculosis
Marwah M. Mohamed a, Shereen A. Fouad a, Hisham A. Elshoky b, Gina M. Mohammed c,
Taher A. Salaheldin b,d,⇑
a
Department of Bacterial Diagnostic Products, Veterinary Serum and Vaccine Research Institute, Egypt
b
Nanotechnology and Advanced Material Central Lab, Agriculture Research Center, Egypt
c
Central Laboratory for Evaluation Veterinary Biologics, Agriculture Research Center, Egypt
d
Mostafa Elsayed Nanotechnology Research Centre, British University in Egypt, Egypt

a r t i c l e i n f o a b s t r a c t

Article history: Corynebacterium pseudotuberculosis is the etiological agent of chronic caseous lymphadenitis. The bac-
Received 4 January 2017 terium infects goats and sheep causing great economic loss worldwide annually. The present work aims
Revised 3 February 2017 to evaluate the efficiency of gold nanoparticles (AuNPs) and AuNPs – laser combined therapy as antibac-
Accepted 13 February 2017
terial approaches against C. pseudotuberculosis bacteria in vitro. Gold nanoparticles 25 nm were synthe-
Available online xxxx
sized by co-precipitation method and characterized by different techniques including; Transmission
Electron Microscope (TEM), X-ray Diffraction and Dynamic Light Scattering. Three concentrations of
Keywords:
AuNPs (50, 100 and 200 lg/ml) were utilized for estimating the bacterial growth rate and the
C. pseudotuberculosis
Gold nanoparticles
Minimum Inhibitory Concentration (MIC). The mechanism of interaction between AuNPs and bacteria
Laser was evaluated by transmission electron microscopic image analysis. Confocal Laser Scanning
Antibacterial agent Microscopic technique was used to study the cytotoxic action of AuNPs and laser against C. psudotuber-
Caseous lymphadenitis culosis. Results revealed that MIC of AuNPs and AuNPs – laser combined therapy were 200 lg/ml and
Sheep and goats 100 lg/ml respectively. TEM image analysis illustrated that gold nanoparticles penetrated the thick wall
of C. psudotuberculosis and accumulated as intracellular agglomerates. Laser light enhanced the antimi-
crobial activity of gold nanoparticles by at least one fold due to its photo thermal combined effect that
might be used as an effective antibacterial approach against C. pseudotuberculosis.
Ó 2017 Faculty of Veterinary Medicine, Cairo University. Production and hosting by Elsevier B.V. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Most of the therapeutic routs are futile even the chemothera-
peutic one that is because of the thick capsule that surrounds the
Caseous lymphadenitis (CLA) is one of the most sporadic abscesses of C. pseudotuberculosis [4]. Although the vaccination
chronic bacterial origin disease, infects mainly goats and sheep against C. pseudotuberculosis with dead bacteria or with an
caused by C. pseudotuberculosis. This organism is gram positive, excreted proteins provides limited protection, the need for anti-
non-spore and facultative anaerobic rod shaped bacteria, several genic compounds that can activate both humeral and cellular arms
of which are pathogenic for man and animals. As long as the animal of the immune system is still the demand [5]. However, such meth-
becomes infected with C. pseudotuberculosis, it survives and repli- ods are hard to achieve and hence limited effectiveness.
cates within cells of the immune system that normally be the Nanomaterials became a promising and efficient candidate that
defense system against it and once the disease established, CLA can replace conventional materials with most applications in all
is difficult to eradicate [1–3]. fields of science and technology. As a result of the ultra-small size
of nanomaterials that have higher surface to volume ratio and
increased number of active atoms at their outer surfaces [6]. Some
Peer review under responsibility of Faculty of Veterinary Medicine, Cairo Univer-
metallurgic nanomaterials have been approved as bactericidal and
sity.
⇑ Corresponding author at: Mostafa Elsayed Nanotechnology Research Centre, bacteriostatic agents, among those used are silver, gold and zinc,
British University in Egypt, El Sherouk City, and Suez Desert Road, Cairo 11837, P.O. each with different properties and spectrum activities [7,8]. Gold
Box 43, Egypt. nanoparticles (AuNPs) are widely used in enormous biological
E-mail addresses: [email protected], [email protected] (T.A. Salahel- applications mainly in medical and gene therapy and biosensors
din).
for diagnosis. AuNPs are easy to prepare by co-precipitation
URL: http://www.bue.edu.eg (T.A. Salaheldin).

http://dx.doi.org/10.1016/j.ijvsm.2017.02.003
2314-4599/Ó 2017 Faculty of Veterinary Medicine, Cairo University. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Mohamed MM et al. Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis. Int J of Vet Sci
Med (2017), http://dx.doi.org/10.1016/j.ijvsm.2017.02.003
2 M.M. Mohamed et al. / International Journal of Veterinary Science and Medicine xxx (2017) xxx–xxx

approach and may have lower toxicity compared to other metallic the precipitated reddish brown pellet was dried in vacuum oven
nanomaterials such as silver nanoparticles [9]. The problem of for 2 h then grinded into fine powder to be bombarded by X-ray
antibiotic resistant bacteria and their emphasis on health care for phase analysis. The corresponding XRD pattern was recorded
costs, that encourages researchers to innovate new approaches to in the scanning mode (X’pert PRO, PAN analytical, Netherlands)
develop more effective antimicrobial agents to overcome the bac- operated by Cu K radiation tube (=1.54 A°) at 40 kV and 30 mA.
terial resistance and reduce their cost [10]. One approach being The obtained diffraction pattern was interpreted by the standard
tested is photodynamic therapy (PDT), which uses light absorbing ICCD library installed in PDF4 software. Qualitative and quantita-
dyes to generate toxic oxygen radicals to kill the bacteria. How- tive measurements of the applied gold nanoparticles concentra-
ever, this treatment might not be effective for infections in hypoxic tions were determined by Inductivity Coupled Plasma (ICP)
environments. Another promising approach is to use metal technique (PerkinElmer ICP-OES: Optima 2000, Germany). Synthe-
nanoparticles, and laser energy to physically damage the bacteria sis and characterization of gold nanoparticles were performed in
through Photo Thermal Therapy (PTT) [11]. The optical properties Nanotechnology & Advanced Materials Central Laboratory, Agricul-
of conductive nanoparticles (NPs), such as those made of gold have ture Research Center, Egypt.
been associated with the Surface Plasmon Resonance (SPR), which
when confined to small colloids, is referred to as the localized sur- 2.3. Strain and growth culture
face Plasmon resonance (LSPR). This phenomenon, in which the
surface electrons oscillate collectively when irradiated with partic- For study the antibacterial activity of Au NPs on C. pseudotuber-
ular light energy resonated with its LSPR, causes wavelength culosis, a local strain was isolated from lymph nodes of a native
dependent photo thermal effect. When gold NPs absorb resonating sheep suffered from caseous lymphadenitis, it was identified mor-
light energy, they release heat in accordance, making them useful phologically, biochemically and biologically at Bacterial Diagnostic
in photo thermal therapy applications such as targeting cancer Products, Veterinary Serum and Vaccine Research Institute, Cairo,
and bacterial cells. Laser-induced photothermal phenomena Egypt. Brain heart infusion broth (DIFCO, Detroit, Mich., USA) and
induce physical disruption of the bacterial cells leading to death Brain heart infusion agar (DIFCO, Detroit, Mich., USA) were used
[12]. The purpose of this study is to evaluate the efficiency and as culture media [14,15]. The growth culture was performed using
mechanism of the antimicrobial activity of gold nanoparticles single colony of fully identified C. pseudotuberculosis bacteria field
(AuNPs) and AuNPs–laser combined therapeutic approach against strain, it was picked up and inoculated into 100 mL of brain heart
C. pseudotuberculosis bacteria. infusion broth then incubated at 37 °C for 48 h. Serial dilutions
were carried out using broth media to give a final organism density
of 105 CFU/mL.
2. Materials and methods
2.4. The antimicrobial activity of AuNPs
2.1. Synthesis of gold nanoparticles (AuNPs)
The Minimum Inhibitory Concentration (MIC) of three different
Gold nanoparticles colloidal solution (25 ± 5 nm) was synthe-
concentrations of AuNPs (50, 100 and 200 mg/mL was determined
sized by co-precipitation protocol through the reduction of Gold
using the plate count method [16,17]. Twenty mL from the previ-
Chloride hydrate (HAuCl4) (99.99%, Aldrich, USA) with Sodium
ously prepared culture medium (containing 105 CFU/mL of bacte-
citrate tribasic dihydrate (99%, Aldrich, USA) Sunder boiling condi-
rial cells) was a liquated into four tubes (5 mL each), then the Au
tions [13]. All glass wears were cleaned and sterilized by aqua regia
NPs were added in each tubes in the following concentration 0,
and dried in oven dryer at 120°C. DNA free deionized water (Milli-
50, 100 and 200 mg/mL, respectively. The tubes were incubated in
pore, USA) was used for preparation and dilutions. Fifty mL
a shaking incubator at 37°C for 24 h. After incubation, 50 mL from
(0.03 mM) HAuCl4, in 250 mL beaker, was brought to boil under
each tube was spread onto brain heart infusion agar and incubated
stirring for 5 min. Sodium citrate tribasic dihydrate (0.5 mL) 1%
at 37°C for 24 h; the numbers of colonies growing on agar were
solution was added at once under continuous stirring. The solution
estimated. Measurements of the optical density (O.D.) at 600 nm
color turned bright red forming gold nanoparticles colloid, then left
of the treated bacteria were graphed to estimate the growth curves
to cool and proceed for physiochemical characterization.
[10].

2.2. Characterization of gold nanoparticles 2.5. Laser induced gold nanoparticles antibacterial effects

The characteristic SPR of AuNPs was recorded by absorption The MIC for 3 groups was performed from a mixture of culture
spectroscopic technique using a double beam UV–Vis-NIR spec- media and Au NPs (0, 50, 100 and 200 mg/mL) [16,17], the culture
trophotometer (Cary 5000, Agilent, UK) within the scanning range tubes were incubated in a shaking incubator at 37°C for 24 h.
of 200–800 nm. Actual morphology of the prepared gold nanopar- Incubation tubes were subjected to 520 nm laser light, 20 mW, at
ticles was imaged by High Resolution Transmission Electron different exposure time (5, 10 and 20 min.) for each group,
Microscope (HR-TEM) operating at an accelerating voltage of respectively. Fifty mL from each culture tube were spread onto
200 kV (Tecnai G2, FEI, Netherlands). Diluted colloidal gold brain heart infusion agar and incubated at 37°C for 24 h; the
nanoparticles solution was ultra-sonicated for 5 min to reduce numbers of colonies growing on agar were estimated. Measure-
the particles aggregation. Using micropipette, three drops from ments of the O.D. at 600 nm of the treated bacteria were graphed
the sonicated solution were deposited on carbon coated-copper to estimate the growth curves [10].
grid and left to dry at room temperature. HR-TEM images of the
gold nanoparticles that deposited on the grid were captures for 2.6. AuNPs antibacterial mode of action
morphological evaluation. Dynamic Light Scattering (DLS) tech-
nique was utilized to estimate the average particle size distribution Electron microscope imaging and EDX analysis were performed
that was measured by zeta sizer (Malvern, ZS Nano, UK). The to study the effect of AuNPs on the bacterial cell integrity and to
chemical structure of prepared gold nanoparticles was assessed elucidate whether the mode of action either extra or intra cellular.
using X- ray Diffraction (XRD) technique. Colloidal gold solution Culture treated with different concentrations of AuNPs and non-
was centrifuge at 18,000 rpm for 30 min using cooling centrifuge, treated one of C. pseudotuberculosis were proceeded for fixation

Please cite this article in press as: Mohamed MM et al. Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis. Int J of Vet Sci
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 M.M. Mohamed et al. / International Journal of Veterinary Science and Medicine xxx (2017) xxx–xxx 3

process and sectioned as the following; bacteria were fixated in fix- sents the particle size distribution curve obtained from DLS mea-
ation solution (2% glutaraldehyde + 2% paraformaldehyde dis- surements with excellent polydispersity index (PDI) 0.106. The
solved in 0.12 M Sorensen’s buffer, pH 7.4) and then stained by measurement of the gold nanoparticles surface charge, zeta poten-
(2%) Osmium Tetroxide solution for 1 h at 4°C. Embedding sections tial, was ( 39 mV) as measured by DLS technique. Gold nanoparti-
were cut on a MT6000 ultra microtome (Sorvall, New Castle, DE). cles phase formation was tested by XRD analysis, Fig. 1D, based on
Thin sections (0.1–0.05 mm) were examined and photographed Bragg’s reflections low. Characteristic diffraction pattern showed
using electron microscope (Tecnai G2, FEI, Netherlands) [11]. sharp intense and narrow peaks at 38.14°, 44.39°, 64.61°, 77.58°
and 81.13° 2h angles those corresponding to hkl parameters of
2.7. Confocal Laser Scanning image (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2), respectively. The obtained
diffraction pattern was compared with the standard ICCD library
Confocal Laser Scanning Microscopic (CLSM) (710, Carrel Zeiss, installed in PDF4 software, card no: (04-003-3089).
Germany) was used to study the antibacterial action of AuNPs on
C. pseudotuberculosis bacteria at gold concentrations of 50, 100 3.2. Antibacterial activity of AuNPs/AuNPs-laser enhanced
and 200 mg/mL, for discrimination of live from dead cells on the
basis of membrane integrity. Acridine Orange fluoresces green The MIC of AuNPs against C. pseudotuberculosis was 200 mg/mL
(AO) (sigma-Aldrich, USA) was used to stain live cells while Ethid- Fig 2. While the MIC of AuNPs-laser Enhanced was recorded to
ium Bromide fluoresces red (EB) (sigma-Aldrich, US) was used to be 100 mg/mL. Fig 2 illustrates a comparative study between the
stain dead cells. In brief, 20 mL of C. pseudotuberculosis bacteria uses of AuNPs alone as antibacterial active agent against C. pseudo-
before and after treatment (AuNPs and Laser) were placed in glass tuberculosis bacteria and AuNPs-laser combined therapy. Laser
bases plates and 5 mL of AO/EB (1 mg/mL, 0.3 mg/mL, respectively) enhancement depended upon the exposure time. For dose of
were added and left for 5 min, then the samples have been exam- 50 mg/mL AuNPs, the inhibition of the bacterial growth was 75%,
ined immediately under confocal microscopy before leakage of 85% and 95% for exposure time of 5 min, 10 min and 20 min,
dyes from cells after five minutes. Microscopic inspection of the respectively, as clearly recorded in the corresponding growth
tested bacteria was carried out using the excitation laser lines at curves.
405/530 and 543/640 nm [18].
3.3. AuNPs antibacterial mode of action
3. Results
The morphological changes of bacterial cells represented infor-
3.1. Characterization of gold nanoparticles mation about the antibacterial mode of action of gold nanoparti-
cles either extracellular or intracellular. High Resolution
Fig. 1 shows physicochemical characterization of the synthe- Transmission Electron Microscope (HRTEM) imaging of AuNPs
sized gold nanoparticles to evaluate its properties using different and AuNPs-laser treated C. pseudotuberculosis bacteria compared
techniques. Colloidal solution of spherical gold nanoparticles had to the untreated control one. Fig 3A shows HR-TEM image of trea-
a distinct bright red color and corresponding characteristic Surface ted bacteria, it was observed that gold nanoparticles were capable
Plasmon Resonance (SPR) absorption peak at 524 nm with Gaus- to penetrate the thick cell wall of C. pseudotuberculosis bacteria
sian distribution curve, as indicated in Fig. 1A. For nanoparticle suggesting the intracellular mechanism of action. Energy Disper-
morphology and size determination, HR-TEM electrograph showed sive X-ray (EDX) analysis of the imaged sample confirmed the for-
well uniformed spheres with average size of 25 ± 5 nm. The corre- mation of gold aggregates inside the cytoplasm as shown in Fig 3B.
sponding TEM diffraction pattern inset showed formation of gold The cell wall of control group was appeared intact, however, vac-
nanoparticles with cubic crystal structure, Fig.1B. Fig. 1C repre- uoles formed inside the bacterial cells treated with AuNPs, while

Fig. 1. Characterization of gold nanoparticles (AuNPs). (A): Absorption spectrum of gold nanoparticles. (B): HRTEM image showing spherical shape of prepared gold
nanoparticles with average size 25 nm. (C): Particle size distribution of prepared gold nanoparticles showing the average size of 25 nm. (D): XRD pattern analysis indicating
the formation of gold nanoparticles with cubic unit crystal.

Please cite this article in press as: Mohamed MM et al. Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis. Int J of Vet Sci
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Fig. 2. Antibacterial activity of AuNPs and Laser induced AuNPs against C. pseudotuberculosis at different gold concentration (0. 50, 100 and 200 mg/ml) and different laser
exposure times (5, 10 and 20 min). Plates represent the plate count method describing the number of growth colonies and the growth curves represent the percentage of
inhibition rate of all treatments.

Fig. 3. HR-TEM electrograph of intracellular localization of gold nanoparticles within C. pseudotuberculosis bacteria, (A): penetration of AuNPs into the cell wall and
accumulation in cytoplasm by HR-TEM. (B): Energy Dispersive X-ray (EDX) image analysis of the intracellular AuNPs aggregates.

complete destruction was recorded within bacteria exposed to With the same extent, CLSM imaging of the combined treatment
combined AuNPs–Laser treatment. As clearly illustrated in Fig 4. (AuNPs-Laser), Fig. 6 represents the distribution of dead/viable bac-
teria in the AuNPs-Laser (5 min laser exposure) co-culture tested
concentrations (0, 50 mg/mL, 100 mg/mL and 200 mg/mL AuNPs).
3.4. Confocal Laser Scanning Microscopic imaging
Where in Fig. 6A, the control untreated live bacteria appeared in
green fluorescence. Fig. 6B represents the 50 mg/mL AuNPs-Laser
Confocal Laser Scanning Microscopic (CLSM) imaging of C. pseu-
treatment, the viability was about 50% that is why yellowish orange
dotuberculosis (treated with both AuNPs and AuNPs-Laser) stained
fluorescence due to the cross over between the green (live cells) and
with Acridine orange (AO) fluoresces green (stain life cells) and
red (dead cells). The viability was zero for concentrations of
Ethidium bromide (EB) fluoresces red (stain dead cells) which used
100 mg/mL and 200 mg/mL AuNPs-Laser, where red fluorescence
for visual differentiation between live and dead cells based on
appeared as a result of dead abundance, Fig. 6C and D. It is remarked
membrane integrity. Fig. 5 represents the distribution of dead/
from Fig. 6D that laser disintegrates the bacterial cells as a low
viable bacteria in the gold nanoparticles co-culture tested concen-
number of dead cells appear.
trations (0, 50 mg/mL, 100 mg/mL and 200 mg/mL AuNPs), where in
Fig. 5A, only live bacteria appear in green fluorescence which is
corresponding to the control untreated bacteria. Fig. 5B represents 4. Discussion
the 50 mg/mL AuNPs treatment, the viability was about 50% that is
why yellowish orange fluorescence due to the cross over between Nanotechnology has attracted global attention because
the green (live cells) and red (dead cells). The viability was zero for nanoparticles have novel and unique properties from their bulk
concentrations of 100 mg/mL and 200 mg/mL AuNPs, only red fluo- equivalents. The antibacterial activity of nanoparticles largely has
rescence appeared as a result of dead abundance, (Fig. 5C and D). been studied with human pathogenic bacteria, such as E. coli and

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Fig. 4. Intracellular mode of action imaged by HR-TEM. (A): untreated bacterial cell. (B): AuNPs induce vacuole formation in cytoplasm. (C): AuNPs-laser induced cellular
destruction.

Fig. 5. CLSM images for viable/dead C. pseudotuberculosis bacteria in the AuNPs co-culture and stained with Acridine Orange (AO) green fluorescence and Ethidium Bromide
(EB) red fluorescence.. (A) Deionized Water control; (B) 50 lg/mL AuNPs. (C) 100 lg/mL AuNPs; (D) 200 lg/mL AuNPs. Magnification, 40.

S. aureus [10]. It has been reported that the biophysical interactions ture analysis, XRD pattern confirms the phase formation of gold
occur between NPs and bacteria through biosorption, aggregation, nanoparticles with face-centered cubic crystal structure.
and cellular uptake causing membrane damage and toxicity. Still a The MIC is relatively high compared to previous published work
comprehensive understanding of antibacterial mechanisms is that studying the bactericidal effect of gold nanoparticles against
needed to improve the effectiveness of NPs in disease treatment the different bacterial stains such as K. pneumoniae, S. typhi, P.
[8]. aeruginosa and E. coli, where MICs ranged from 20 to 40 mg/mL.
In this investigation, the antibacterial effects of AuNPs on C. the reason might be attributed to the thicker wall of C. pseudotu-
pseudotuberculosis was studied. Gold nanoparticles were synthe- berculosis bacteria that reduces the penetration rate of AuNPs
sized using citrate reduction method that is an easier and rapid through cell wall and hence reduces the antibacterial activity of
method with controlled size and shape [13]. The shape absorption AuNPs at lower concentrations. It is recorded that, the laser light
peak with well performed Gaussian distribution implies uniformity enhances the antimicrobial activity of gold nanoparticles by at
of gold nanoparticles formation with no aggregation and perfect least one fold. This might be attributed to the photothermal effect
dispersion [19,20]. The average size of the synthesized AuNPs resulting from the unique property of nano scaled gold colloidal
was 25 ± 5 nm with spherical shape as illustrated by high resolu- solution that has strong enhanced absorption band at 524 nm cor-
tion electron microscopic image and DLS measurement. The Low responding to the AuNPs Surface Plasmon Resonance (SPR) oscilla-
Polydispersity Index (PDI), 0.106, reflects monodispersity and per- tions. Upon exposure to resonating laser emission line, the kinetic
fect dispersion of the nanoparticles with no aggrigation. The high energy of gold nanoparticles increases resulting in local heating of
electronegative value ( 39 mV) of zetal potential indicates high the exposed target which is the gold nanoparticles treated bacteria
degree of gold nanoparticles collidal stability and perfect capping in the present experiment. The generated photothermal effect can
effect of citrate ions and particle-particles repulsion that prevents be employed for rapid and efficient bacterial cell destruction
the particles aggregation or precipitation [21]. For chemical struc- [22–25]. Laser enhancement depends on the exposure time.

Please cite this article in press as: Mohamed MM et al. Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis. Int J of Vet Sci
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Fig. 6. CLSM images for viable/dead C. pseudotuberculosis bacteria in the AuNPs co-culture and stained with Acridine Orange (AO) green fluorescence and Ethidium bromide
(EB) red fluorescence, staining. (A) Laser - deionized Water control; (B) 50 mg/mL AuNPs-Laser. (C) 100 mg/mL AuNPs-Laser; (D) 200 mg/mL AuNPs-Laser. Magnification, 40.

Similar results were obtained from other studies, which showed combined approach, AuNPs-laser, causing quick loss of cell mem-
that the laser enhanced nanoparticles applied to S. aureus reduced brane integrity. The laser light enhances the antimicrobial activity
significantly the number of viable bacteria [26]. Zharov et al. [11] of gold nanoparticles by at least one fold. It could be concluded that
applied the combined triple approach, gold nanoparticles, laser the AuNPs combined with laser exposure be used as local effective
and multifunctional photothermal microscope, for localized killing antibacterial approach inhibits the growth of C. pseudotuberculosis.
of S. aureus in vitro where photothermal bacterial damage accom-
panied by formation of bubble due to local overheating produced
by gold nanoparticles upon absorbing the incident laser light Disclosure
energy, that transforms directly into heat energy [11].
The morphological changes of bacterial cells represent informa- The authors of the present work report that there is no conflict
tion about the antibacterial action mode of gold nanoparticles of interest in this work.
either extracellular or intracellular that imaged by HRTEM and
by EDX. The suggested mechanism for antibacterial activity of
the gold nanoparticles is attributed to generation of Reactive Oxy- References
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Please cite this article in press as: Mohamed MM et al. Antibacterial effect of gold nanoparticles against Corynebacterium pseudotuberculosis. Int J of Vet Sci
Med (2017), http://dx.doi.org/10.1016/j.ijvsm.2017.02.003