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International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 1
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
Antimicrobial and Antibiofilm Activity of Ag and
Ni Nanoparticles Against Some Bacterial Pathogens Sahar E. Abo-Neima
1 and Eman A. H. Mohamed
2*
1Physics Department, Faculty of Science, Damanhur University, Egypt. 2Botany and Microbiology Department, Faculty of Science, Damanhur University, Egypt.
*Corresponding author: [email protected], phone number: 002 01027737785
Abstract-- Three multi drug resistant human pathogens, Pseudomonas aeroginosa, Staphylococcus aureus and Bacillus
subtilis, have been subjected to different concentrations of Ag
and Ni nanoparticles (NPs). P. aeroginosa was the most
stubborn pathogen that resisted even Ni. One mg/ml of Ag-NPs
was efficient to affect its growth dramatically. However, the
lowest used concentrations of Ni NPs (10-4mM) and Ag-NPs
(10-3mM) have strong antimicrobial activity against the other
two pathogens, S. aureus and B. subtilis. Growth inhibition %
was also calculated regarding absorbance readings of the
pathogenic cells in the presence of the NPs. The inhibition %
was increased with NPs concentration and reached its
maximum values with the highest concentration of Ag-NPs
(1mg/ml). Antibiofilm activity of Ag and Ni-NPs was also tested
in this work against the three bacterial pathogens. One mg/ml
Ni NPs reduced cells OD600 of P. aeroginosa from o.35 to 0.14.
Ag NPs were more effective antibiofilm agent and 1 mg/ml of
them reduced Pseudomonas growth from 0.48 to 0.02.
However, Ag is more effective against the pathogenic biofilm
than Ni for the other pathogens too. Nanoparticles were
characterized using both scan and transmission electron
microscopy. Results revealed that NPs are spherical and have
sizes ranging from 10 to 25 nm for Ag and 25- 82 nm for Ni.
Different shapes of cell distortions were noticed when
pathogens treated with the NPs such as cell membrane
detachment, cell elongation, shrinking and leakage of cell
content. According to the antimicrobial and antibiofilm
activity of Ag and Ni NPs against important pathogens, we
recommend to utilize these promising NPs in many
applications such as medical devices, water sanitization and wound dressing.
Index Term-- Ag and Ni nanoparticles, antimicrobial, antibiofilm, TEM
INTRODUCTION
Many bacterial infections are treated by antibiotics because of their powerful effect on pathogens. However, the
widespread of antibiotics use has led to the appearance of
multidrug-resistant bacterial strains. These bacteria are
resistant to nearly all antibiotics. There are three major
targets of antibiotics in bacterial cells: translation
machinery, DNA replication and cell wall synthesis.
Bacterial resistance can develop against any of these modes
of action [1]. Nanotechnology considers the production of
materials with at least one dimension (1-100 nm) [2],
however, nanoparticles (NPs) have shown antibacterial
properties against Gram-positive and Gram-negative
bacteria. Fortunately, antibiotic resistance mechanisms are
irrelevant to NPs. This is because NPs mode of action targets to the cell wall directly with no need to bacterial cell
penetration. Accordingly, Attention was focused on
materials based on nanoparticles with antibacterial effect
[1]. Moreover, NPs act also as a carrier of antibiotics. They
increase antibiotics serum levels and inhibit bacterial
resistance [3]. Therefore, nanoparticles are promising and
can replace conventional materials in many applications due
to their ultra- small size and high surface to volume ratio
[4]. Antimicrobial activities of metal NPs like Ag, Ni, Co,
and Cu have been previously reported [5]. Silver is an
antimicrobial metal that is widely used for sterilization
purposes including medical devices and water sanitization [6]. Ag NPs have concentration dependent antibacterial
activity against strong pathogens such as Escherichia coli
and Pseudomonas aeruginosa. [1]. Accordingly, Ag NPs are
widely used in antibacterial coating of implantable devices,
bone cement, dental materials, wound dressing and other
applications [7-10]. Moreover, Ag NPs prevent biofilm
formation by inhibition of expression of some bacterial
genes [11]. Accordingly, they have an effect on both the
developing and matured biofilms [12].
Ni NPs were also reported in some studies as
antimicrobial agents [13-16]. NiO nanoplates for example
showed zones of inhibition against important pathogens
such as Bacillus subtilis, Staphylococcus aureus,
Escherichia coli and Pseudomonas vulgaris [17]. Pandian et
al, 2016 [18] revealed that Ni NPs have effective
antimicrobial activity at 50 µl/ml and therefore can be used
as antimicrobial coatings of materials for environmental and
medical applications. Moreover, Suitable concentrations of
Ni NPs can reduce the biofilm produced by Staphylococcus epidermidis greatly [19].
NPs have to be accurately characterized due to the
variety of methods available for their synthesis. NPs
chemical and physical properties are always can be related
to their behaviour in biological systems. Therefore, at least
simple characterization (size and shape) should be achieved.
The most common used methods for simple NPs
specification are achieved by direct imaging and measurement. This can be done by electron microscopy,
mailto:[email protected]
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 2
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
UV-Visible Spectroscopy, and dynamic light scattering [20-
22].
In this study, three different bacterial human
pathogens (Staphylococcus aureus, peusodomonas
aeroginosa and Bacilus subtilis) have been subjected to
growth inhibition using antibiotic discs, Ni nanoparticles of
different concentrations, and various dilutions of Ag NPs.
Moreover, antibiofilm effect of these nanoparticles against
the pathogens was also tested. Besides, Ni and Ag NPs were
prepared and characterized using Scan and Transmission
Electron microscopy. Bacterial cells were also examined
and imaged using Transmission Electron Microscopy to
detect different cell distortions after treatment with Ag and
Ni nanoparticles.
MATERIALS AND METHODS
Bacterial strains and growth curves
Bacterial strains used in this study were obtained from
EMCC (Egyptian Microbial Culture Collection),
Department of Food Science, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. These strains are
Pseudomonas aeroginosa ATCC27853, Bacillus subtilis
ATCC21332, and Staphylococcus aureus ATCC25923. All
strains were grown and activated in nutrient broth medium
at 35°C (±2) over night. All growth curves were plotted
between growth absorbance at 600 nm and time in hour [23]
using nutrient broth medium at 35°C (±2). The growth
curves included the growth of every single strain in the
presence and absence of Ag and Ni NPs. Besides, growth
inhibition percentage was calculated according to the
following equation:
Inhibition % =[(C-T)/C] ×100
Where C= O.D.600 of control sample
T= O.D.600 of NPs treated sample
Synthesis of silver nanoparticles by the reduction method
Silver nanoparticles were synthesised by heating 500 ml of
1M AgNO3 (Lobachemie, India) until the boiling stage.
Then, 1% of sodium citrate (MERCK, Germany) was added
dropwise to the boiling solution. The colour of the boiling
mixed solution turned slowly into greyish yellow, representing the reduction of the Ag+ ions. Later on, the
solution was cooled at room temperature and poured into
dark bottle until usage [24].
Preparation of nickel nanoparticles
Ni-NPs were purchased from Sigma-Aldrich, in the form of
nano powder, suspended in TSB (Tryptic Soy Broth, Merck)
and ultrasonicated for 2 h before use. The average size of
particles was less than 100 nm.
Antibiotic and nanoparticles sensitivity test
Cell suspensions from overnight growth in nutrient broth
(O.D600 ~ 0.8) were inoculated in Muller Hinton Agar
(Sigma-Aldrich). Antibiotic discs were then applied to the
inoculated plates and incubated at 35°C (±2) for 24 h. The used antibiotics were: Penicillin (P10), Gatifloxacine
(GAT5), Cefuroxime (CXM30), Tobramycine (TOB10),
Cefsulodin (CES10), Ofloxacin (OFX5), Chloramphenicol
(C30), Rifampin (RA5), Tetracycline (TE30), and Ceftazidime
(CAZ30). Bacterial sensitivity was verified by measuring the
inhibition zones diameters in mm.
For sensitivity against Ag and Ni NPs, dilutions (10-1, 10-2, 10-3, and 10-4) were prepared from 1mM initial
Ni or Ag concentration. Discs for each dilution were applied
to Muller Hinton Agar plates inoculated with the pathogenic
bacteria using the diffusion method. Cultures were then
incubated at 35°C (±2) for 24 h and diameters of inhibition
zones were measured in mm.
Antibiofilm activity of the nanoparticles
Inhibitory effect of Ag and Ni NPs against biofilms formed
by the bacterial pathogens was tested in vitro using the
commonly used 96 wells polystyrene plate method. 10 µl
activated culture of each tested strain (about 106 CFU/ml)
were added. 20 µl of each single NPs were added per each
well. The total volume in each well was adjusted to 250 µl
using Tryptic Soy Broth (TSB). Control wells contained
culture medium and the tested strain without adding the
NPs. After incubation at 35°C for 24 hours, content of the
plates were poured off and the wells were washed three
times with 300 µl of phosphate buffered saline (PBS, pH 7.2). The remaining adhered bacteria were fixed with 250 µl
of methanol per well. After 15 min, plates were emptied and
air dried. The plates were stained with 250 µl per well of
1% crystal violet used for Gram staining for 5 min. The
excess of stain were rinsed off by placing the plates under
running tap water. After drying the plates, absorbance at 600
nm was measured using ELISA reader [25]. All tests were
carried out in triplicate (n=3) and the results were averaged.
Transmission and Scan Electron Microscopy
Morphological changes of NPs treated bacterial cells have
been detected using Transmission Electron Microscopy
(TEM) at TEM unit, Faculty of science, Alexandria
University. Before examination, samples were fixed in
300µl glutaraldehyde and 3% of 0.1 M phosphate buffer
saline, pH=6. Procedure steps were followed according to
AyseInhan et al, 2011 [26]. Samples were examined using
Philips EM201 80 kv Transmission Electron Microscope.
Scan Electron Microscopy SEM and TEM were also employed (Faculty of science, Alexandria University) to
examine the shape and size of Ni and Ag nanoparticles.
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 3
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
RESULTS AND DISCUSSION
Pathogens under test, Staphylococcus
aureus,peusodomonas aeroginosa and Bacilus subtilis, are
generally resistant to most of the used antibiotics, especially
Pseudomonas which is resistant to all of them (Table 1). GAT5 and OFX5 are the most effective antibiotic against B.
subtilis and S. aureus followed by RA5. On the other hand,
all of the tested pathogens are resistant to CXM30 and
CAZ30. Gram-negative bacteria are the main cause of
nosocomial infections and many other diseases. For
example, genetic modifications are rapid in P. aeroginosa
and therefore it resists many antibiotics. Besides, it can
survive harsh environmental conditions [27, 28]. Moreover,
multiple resistance mechanisms are present in a single host
and this eventually leads to multidrug resistance [29, 30].
Mechanisms of resistance in Gram-positive and negative
bacteria include production of enzymes that degrade (such as production of β-lactamase to resist Penicillin) or modify
antibiotics, modification of cell components (such as cell
wall to resist vancomycine and ribosomes to resist
tetracycline), or efflux pumps expressions (leads to multiple
antibiotic resistance) [31, 32]. In conclusion, the abundance
of antimicrobial drugs is no longer effective and bacterial
infections still remain a major issue [1]. Accordingly,
bacterial resistance has become a serious problem that has to
be resolved using new strategies.
Table I
Antibiotic susceptibility against bacterial pathogens. R, resistant
In our study, we used Ag and Ni NPs against the
bacterial pathogens after characterization of the prepared
nanoparticles using (SEM) and (TEM). SEM utilizes a high-
energy electron beam. This beam is scanned over surface
and the back scattering of the electrons is observed [33].
However, SEM is a common technique used to study
morphological and surface characterization, and examine
metal particles size at the nano to micro level scale. This
means that the study area of the sample which can be
viewed in focus at once is quite large [33]. In our study,
SEM revealed that the synthesized silver nanoparticles were sphere-shaped, and their size ranged between 10 and 25 nm
(Fig.1a). Similar results have been obtained by Rajeshkumar
et al, 2019 [33]. Much bigger (25-100nm) and spherical
nanoparticles of Ni were also scanned and showed in Fig.
1b. However, the nanoparticles showed an equal distribution
on the surface. More characterization was performed with
the nanoparticles using Transmission Electron Microscopy
(Fig. 2). The size of spherical silver nanoparticles ranged from 10 to 25 nm (Fig. 2a). Ni nanoparticles (Fig. 2b) were
also spherical but bigger (25- 82 nm).
(a)
(b)
Fig. 1. Scan Electron Microscopy of Ag NPs (a) and Ni NPs (b).
(a)
(b)
Fig. 2. Transmission Electron Microscopy of Ag NPs (a) and Ni NPs (b).
Antibiotic
P. aeroginosa B. subtilis S. aureus
P10
R
R 2.4±0.13
GAT5 2±0.3 3.4±0.32
CXM30 R R
TOB10 1.1±0.06 1.6±0.08
CES10 1.7±0.22 R
OFX5 2.2±0.23 3±0.32
C30 1.9±0.08 R
RA5 1.7±0.15 2.9±0.11
TE30 R 2.9±0.097
CAZ30 R R
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 4
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
Even at low concentration (10-3mM), Ag Nps are
effective against B. Subtilis (inhibition zone is 2.4 mm) and
S. Aureus (inhibition zone is 1.8 mm) (Table 2). As
expected, the inhibition zone diameter decreases with the
increase in dilution. However, 1mM is the most effective
against the 3 pathogens. Interestingly, P. Aeroginosa is resistant to Ag NPs in concentrations less than 1 mM
although the susceptibility of the other pathogens to lower
concentrations. Ag Nps have a wide spectrum against both
Gram positive and Gram negative bacterial isolates. They
increase the antibiotics sensitivity by coating with different
antibiotics such as chloramphenicol, tetracycline, penicillin,
gendamycin, chloramphenicol, streptomycin, ciprofloxacin
and anamycin [34]. Ag Nps disturb the bacterial cell wall
and cause inhibition of DNA replication and therefore
control bacterial growth. They proved a great breakthrough
in the field of nano medicine [34]. In our study, we found
that Ag Nps are effective against three important pathogens. The main advantage of Ag Nps against pathogens is that
Ag+ is relatively nontoxic to human and animals, and is
very effective against bacteria, fungi, and viruses. Silver
ions bind to thiol groups in cell membranes and enzymes
forming S–Ag bonds. This leads to denaturation and
inhibition of DNA replication [35, 36]. They intercalate
between purine and pyrimidine to disrupt the H bonding
between the base pair in the antiparallel strands causing
DNA denaturation and prevent replication [37]. Salomoni et
al, 2017 [38] stated that the antimicrobial activity of 5 µg/
mL AgNPs against two hospital strains of P. aeruginosa was very effective. Moreover, the cytotoxicity evaluation
revealed that up to 2.5 µg/mL of AgNPs are very safe for all
of the tested cell lines.
Table II
Pathogens sensitivity against dilutions of Ag-NPs. R, resistant.
.
B
Beside AgNPs, Ni NPs were also used in this study
against the bacterial pathogens (Table 3). Interestingly, the
lowest used concentration of Ni NPs (10-4mM) has
antimicrobial activity against the tested pathogens except P.
aeroginosa. This indicates the strong persistence of this
multi-drug resistant pathogen. It resists even high Ni NPs
concentration (10mM) whereas the other pathogens showed
sensitivity towards all of the used concentrations. The most
sensitive bacterium was S. aureus followed by B. subtilis.
Results revealed that both nanoparticles have antimicrobial activity against the tested pathogens expect for P.
Aeroginosa that persists all of the used Ni NPs
concentrations. NiNPs showed potential antimicrobial effect
against Klebseilla pneumonia, E. coli, S. aureus, and B.
cereus [39]. Besides, Hafshejaniet et al, 2018 [40], revealed
that the treatment with Ni nanoparticles was very effective
against S. aureus and E. coli. The recommended daily intake
of Ni is about 100 mg. Therefore, the daily dose of nickel will not only increases iron absorption and treats weak bones,
but also will inhibit the growth of some important bacterial
pathogens [41]. Moreover, Ni NPs can be used as antimicrobial
coatings for environmental purposes [40]. Pandian et al, 2016
[18], declared that Ni NPs antimicrobial activity can also be
enhanced when synthesized using leaf extract of Ocimum
sanctum. Moreover, Vahedi et al, 2017 [19], were the first to
investigation the inhibitory effects of Ni NPs on biofilm
production by S. epidermidis. In our study, Ni NPs failed to
inhibit the growth of the multi-drug resistant P. aeroginosa,
despite its effectiveness against the other tested pathogens.
However, Ag NPs were able to inhibit the bacterial growth according to the dilution.
Ag Nps are more effective against the tested
pathogens than Ni NPs, especially for the stubborn
pathogen, P. aeroginosa (tables 2 and 3). Hence, we
selected Ag Nps for more investigation against the three
pathogens.
Table III
Pathogens sensitivity against dilutions of Ni -NPs. R, resistant.
The bacterial growth in presence and absence of
Ag NPs was monitored over time (Fig. 3 a, b and c).
Generally, growth rate was decreased with the increased
concentrations of Ag and the highest growth rate was
recorded in the absence of the nanoparticles. On the other
hand, the lowest growth rate was detected when 1 mg/ml of
Ag NPs was used. Interestingly, growth rates of P.
aeroginosa in the presence of 0, 0.1, 0.01 and 0.001 mg/ml
Ag are so close to each other and only 1 mg/ml Ag was
effective against this pathogen (Fig. 3c). These results are
parallel with those in table 2. P. aeroginosa resisted all the used Ag concentrations except 1 mg/ml. Similar results had
been stated by Salomoni et al, 2017 [38]. They revealed the
strong antimicrobial activity of AgNPs against two hospital
strains belong to P. aeruginosa. They also revealed that up
to 2.5 µg/mL of AgNPs are very safe for all of the tested
cell lines. We strongly recommend the use of AgNPs
Ag NPs in mM P. aeroginosa B. subtilis S. aureus
1 2.1±0.36 3.2±0.28 2.4±0.28 10-1
R 2.8±0.14 2.3±0.13
10-2 2.7±0.12 2.1±0.11 10-3 2.4±0.23 1.8±0.19
Ni NPs in mM
P. aeroginosa B. subtilis S. aureus
10
R
2±0.22 2.4±0.32
1 1.9±0.31 2.3±0.13
10-1 1.6±0.09 2.0±0.26
10-2 1.4±0.13 1.9±0.22
10-3 1.1±0.16 1.3±0.19
10-4 0.9±0.31 0.9±0.08
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 5
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
against bacterial pathogens, especially P. aeruginosa, the
strong multiple drug resistant bacterium.
Fig. 3. Growth curves of S. aureus (a), Bacillus subtilis (b) and P.
Aeroginosa (c) in the presence of different Ag NPs dilutions (1, 0.1, 0.01
and 0.001 mg/ml). Control means no nanoparticles.
Growth inhibition % was also calculated regarding
absorbance readings of the living pathogenic cells in the presence and absence of AgNPs (Fig.4 a, b and c). As
expected, no growth inhibition was recorded in the absence
of the nanoparticles. However, the inhibition % was
increased with nanoparticle concentration and reached its
maximum value with the highest concentration of AgNPs (1
mg/ml). Similar results were declared by Wang et al, 2017
[1].
Fig. 4.Growth inhibition % of S. aureus (a), Bacillus subtilis (b) and P.
Aeroginosa (c) in the presence of different Ag NPs dilutions (1, 0.1, 0.01
and 0.001 mg/ml). Control means no nanoparticles
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 2 4 6 8 10 12 14
Ab
sorb
ance
(6
00
nm
)
Incubation time (hours)
control
0.01mg/ml
0.1mg/ml
1mg/ml
0.001mg/ml
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 2 4 6 8 10 12 14
Ab
sorb
ance
(6
00
nm
)
Incubation time (hours)
control
0.001 mg/ml
0.1mg/ml
1 mg/ml
0.01mg/ml
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 2 4 6 8 10 12 14
Ab
sorb
ance
(6
00
nm
)
Incubation time (hours)
control
0.001 mg/ml
0.01mg/ml
0.1mg/ml
1mg/ml
0
10
20
30
40
50
60
70
80
control 0.001 0.01 0.1 1 Ag NPsmg/ml
Inhibition % 0 27.6 52.1 61.9 77
Gro
wth
inh
ibit
ion
pe
rce
nta
ge
0
10
20
30
40
50
60
70
80
control 0.001 0.01 0.1 1 Ag NPsmg/ml
Inhibition % 0 12.1 22.9 60 71.8
Gro
wth
inh
ibit
ion
pe
rce
nta
ge
0
10
20
30
40
50
60
70
control 0.001 0.01 0.1 1 Ag NPsmg/ml
Inhibition % 0 7.3 12.6 12.6 60.6
Gro
wth
inh
ibit
ion
pe
rce
nta
ge
b
c
a
b
c
a
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 6
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
They stated that Ag NPs have concentration dependent
antibacterial activity against strong pathogens such as
Escherichia coli and Pseudomonas aeruginosa.
Accordingly, Ag NPs are widely used in antibacterial
coating of implantable devices, bone cement, dental
materials, wound dressing and other applications [7-10].
The antibiofilm activity of Ag and Ni nanoparticles
was also tested in this work against the three bacterial
pathogens (Fig.5). Generally, higher nanoparticles
concentrations caused higher antibiofilm activity and
therefore lower absorbance readings were detected.
Although Ni NPs have no effect on P. aeroginosa growth
(Table 2), they have an effect on biofilm formation by this
important pathogen (Fig. 5a). One mg/ml Ni NPs reduced cells OD600 from o.35 to 0.14. Ag NPs were more effective
and 1 mg/ml reduced Pseudomonas growth from 0.48 to
0.02 (Fig. 5b). However, Ag is more effective against the
pathogenic biofilm than Ni for the other pathogens too (Fig.
5c, d, e and f). These results are in agreement with those
stated in tables 2 and 3. Nanoparticles antibiofilm activity
may be due to their ultra-small size, increased surface area
and high biocompetence [12]. Ag NPs damage cell
membrane and cause many cell distortions. Besides, they
penetrate biofilm matrixes. Microbial cells which are
forming biofilms held together by extracellular matrix which contains exo-polysaccharids, proteins, and nucleic
acids [42]. Therefore, this firm biofilm protect
microorganisms from several harsh conditions and
disinfectants [43]. Strong biofilm formers like P.
aeroginosa and S. aureus can resist many drugs by forming
biofilms in body tissues, leading to many infections. In
addition to Ag, suitable concentrations of Ni nanoparticles
can reduce biofilm produced by Staphylococcus epidermidis
greatly [19]. These results match our findings and therefore
both nanoparticles can be used successfully as antibiofilm
agents against pathogenic bacteria.
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
control 0.001mg/ml 0.01mg/ml 0.1mg/ml 1mg/ml
Op
tica
l D
en
sity
( 6
00
nm
)
Concentration of Ni-NPs (mg/ml)
P. aerginosatreated with Ni- NPs
0
0.1
0.2
0.3
0.4
0.5
0.6
control 0.001mg/ml 0.01mg/ml 0.1mg/ml 1mg/ml
Op
tica
l De
nsi
ty (
60
0n
m)
Concentration of Ag-NPs (mg/ml)
p.aerginosatreated with Ag-NPs
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
control 0.001mg/ml 0.01mg/ml 0.1mg/ml 1mg/ml
Op
tica
l D
en
sity
( 6
00
nm
)
Concentration of Ni-NPs (mg/ml)
B.subtilistreated with Ni-NPs
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
control 0.001mg/ml 0.01mg/ml 0.1mg/ml 1mg/ml
Op
tica
l D
en
sity
( 6
00
nm
)
Concentration of Ag-NPs (mg/ml)
B.subtilistreated with Ag-NPs
a
a
)
b
c
d
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 7
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
Fig. 5. Biofilm formation by p.arguinosa (a,b), B. subtilis (c,d) and
S. aureua (e,f) in the presence and absence (control) of Ni and Ag-NPs
respectively.
The interaction between pathogenic cells and the
nanoparticles was recorded using TEM (Fig. 6). The normal
cells, S. aureus, appear dark with smooth membranes (Fig.
6a). This high electron density of the sample means normal
cells [44]. Different shapes of cell distortions can be noticed
when samples were treated with Ag and Ni nanoparticles
(Fig.6). Psedumonas aeroginisa for example showed distorted physical structure when treated with Ag NPs (Fig.
6 b, c, and d). Cell membrane detachment (b) and elongation
(c) can be obviously noticed. Besides, leakage of various
cell contents is detected (d). Bacillus subtilis showed cell
shrinking (e) and accumulation of nanoparticles around the
cells and on cell surface (f and g). Staphylococcus aureus
showed kidney-shaped cells (h) with detached cell walls (i
and j). This bacterium showed similar reaction when treated
with Ni NPs (k, l, and m). Cell expansion is clearly noticed
in case of B. subtilis when treated with Ni NPs (n, o, and p)
with damaged cell walls and cell content leakage. Generally, light areas can be repetitively noticed due to low electron
density of the treated samples [44]. This means extrusions
of cytoplasmic content and increasing in cell permeability
due to loss of control of transport through cell membranes
[44]. Similar results have been detected by Abo-Neima and
El-Kholy, 2016 [45]. They observed different types of cell
shrinking, leakage, expansion and increasing cell
permeability resulting eventually to cell death when
nanoparticles were applied using bacterial pathogens.
(b) (c)
(d) (e)
(f) (g)
(h) (i)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
0.5
control 0.001mg/ml 0.01mg/ml 0.1mg/ml 1mg/ml
Op
tica
l D
en
sity
( 6
00
nm
)
Concentration of Ni-NPs (mg/ml)
S.aureustreated with Ni-NPs
0
0.1
0.2
0.3
0.4
0.5
control 0.001mg/ml 0.01mg/ml 0.1mg/ml 1mg/ml
Op
tica
l D
en
sity
( 6
00
nm
)
Concentration of Ag-NPs (mg/ml)
S.aureus treated with Ag-NPs
(a)
e
f
International Journal of Basic & Applied Sciences IJBAS-IJENS Vol:19 No:05 8
192405-7676- IJBAS-IJENS @ October 2019 IJENS I J E N S
(j) (k)
(l) (m)
(n) (o)
(p) Fig. 6. TEM of nanoparticles treated and untreated bacterial cells. a,
untreated cells; b, c and d, Ag NPs treated P. aeroginisa cells; e, f and g,
Ag NPs treated B. subtilis cells; h, i and j, Ag NPs treated S. Aureus cells;
k, l and m, Ni NPs treated S. Aureus cells; n, o and p, Ni NPs treated B. Subtilis cells.
CONCLUSION
In our study, three important bacterial pathogens, P.
aeroginosa, S. aureus, and B. subtilis, were found to be
resistant to most of the used antibiotics. On the other hand,
they have found to be sensitive to Ag and Ni NPs in general.
These nanoparticles have both antimicrobial and antibiofilm
activity against the pathogenic bacteria, especially Ag. SEM
and TEM revealed that both metals are nano-sized, spherical
and have the ability to cause many types of distortions to
bacterial cells. Accordingly, we recommend the utilization of these nanoparticles in medical and environmental
applications.
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