Antimicrobial and Antibiofilm Activity of Ag and Ni ...ijens.org/Vol_19_I_05/192405-7676-IJBAS-IJENS.pdfwidely used in antibacterial coating of implantable devices, bone cement, dental

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,

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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.



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



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



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

)


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

)


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


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


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



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


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


Op

tica

l D

en

sity

( 6

00

nm

)


B.subtilistreated with Ni-NPs

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4


Op

tica

l D

en

sity

( 6

00

nm

)


B.subtilistreated with Ag-NPs

a

a

)

b

c

d



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


Op

tica

l D

en

sity

( 6

00

nm

)


S.aureustreated with Ni-NPs

0

0.1

0.2

0.3

0.4

0.5


Op

tica

l D

en

sity

( 6

00

nm

)


S.aureus treated with Ag-NPs

(a)

e

f



(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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Antimicrobial and Antibiofilm Activity of Ag and Ni ...ijens.org/Vol_19_I_05/192405-7676-IJBAS-IJENS.pdfwidely used in antibacterial coating of implantable devices, bone cement, dental