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Jehan et al. World Journal of Pharmaceutical Research

ANTIBIOFILM EFFECT OF IRON OXIDE NANOPARTICLES

SYNTHESIZED BY LACTOBACILLUS FERMENTUM ON CATHETER

Jehan A. S. Salman*1, Mohammed F. Al –Marjani

2, Raghad A. Abdulrazaq

3, Inas A. S

Salman4

and Nawar B. Kamil5

1,2,3

Department of Biology – College of Science – Al- Mustansiriyah University.

4College of Dentistry– Al- Mustansiriyah University.

5College of Dentistry- University of Baghdad.

ABSTRACT

The iron oxide nanoparticles have been synthesized by Iraqi isolate of

Lactobacillus fermentum. Nanoparticles were characterized by Atomic

Force Microscope (AFM) and found the average size 68.28 nm.

Antibacterial activity of the Iron Oxide nanoparticles were investigated

against some pathogenic bacteria by using co culture technique ,the

results showed that iron oxide nanoparticles had inhibitory effect

against pathogenic bacteria with reduction of growth reached to

(46,35,30)% for Staphylococcus aureus, Escherichia coli and Serratia

marcescens respectively. The effect of iron oxide nanoparticles

combined with different antibiotics was investigated against

pathogenic bacteria using disk diffusion method, the results showed the

antibacterial activities of some antibiotics like cephalexin and Nalidixic acid have been

increased in the presence of iron oxide nanoparticle against E.coli. Anti biofilm effect of iron

oxide nanoparticles on coated catheters was observed against S. aureus and E.coli, iron oxide

nanoparticles recorded maximum biofilm inhibition 33.97% against S. aureus, followed by

16.92% occurred against E.coli, While no inhibition on biofilm formation of S.marcescens

was observed.

KEY WORDS: Nanoparticles, Iron oxide, biosynthesis, Lactobacillus, Antibiofilm.

INTRODUCTION

Nanotechnology is applied to various fields such as biological, physical, chemical and

engineering sciences where novel techniques are being developed to probe and manipulate

World Journal of Pharmaceutical Research SJIF Impact Factor 5.990

Volume 4, Issue 8, XXX-XXX. Research Article ISSN 2277– 7105

Article Received on

04 June 2015,

Revised on 28 June 2015,

Accepted on 21 July 2015

*Correspondence for

Author

Jehan A. S. Salman

Department of Biology –

College of Science – Al-

Mustansiriyah University.

marjani20012001@yahoo.com

Jehanmmd@yahoo.com

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Jehan et al. World Journal of Pharmaceutical Research

single atoms and molecules. The metallic have applications in various areas such as coating,

packaging electronics, cosmetics and biotechnology.[1]

Nanoparticles can traverse through the

vasculature and localize any target organ, this leads to new therapeutic, imaging and

biomedical application.[2]

Emergence of new resistant bacterial strains to current antibiotics has become a worldwide

problem and serious public health issue, which raised the need to develop new bactericidal

materials. However, the phenomenon of enhanced biological activity and certain material

changes resulting from nanoparticles is not yet understood fairly. Investigations have shown

encouraging results about the activity of various drugs and antimicrobial formulation in the

form of nanoparticles.[3]

Bacteria get adhered to the biomaterial surfaces and grow to form biofilms. The biofilm mode

of growth protects the bacterial cells against the host defense system and antibiotics.[4]

Iron oxide have received specific attention because of their variety of scientific and

technological applications such as biosensor,[5]

antimicrobial activity,[6]

food preservation,[7]

magnetic storage media, ferrofluids, magnetic resonance imaging, magnetic refrigeration, cell

sorting, targeted drug delivery and hyperthermic cancer treatments.[8]

Besides, it has also

been widely used in biomedical research because of its biocompatibility and magnetic

properties.[9]

Many organisms can produce either intracellular or extracellular inorganic substances.[10]

Bacteria have been most extensively researched for nanoparticles synthesis because of their

fast growth and relative ease of genetic manipulation. The Lactobacillus strains were exposed

to larger concentration of nanoparticles to produced gold,silver and alloy crystals of defined

morphology.[11,12]

Lactobacillus finding its widespread functional prodigality in order to

synthesize nanoparticles of ZnO,[13]

Sb2O3,[14]

BaTiO3 , CdS,[15]

Ag,[16]

and TiO2.[17]

The

present study was conducted to synthesis of iron oxide nanoparticles by locally Lactobacillus

fermentum and study their Antibacterial and Antibiofilm activity.

MATERIALS AND METHODS

Lactobacillus fermentum

Lactobacillus fermentum isolated from Vaginal samples obtained from Department of

biology/College of Science/Al-Mustansiriyah University/Baghdad / Iraq.

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Jehan et al. World Journal of Pharmaceutical Research

Pathogenic bacteria

Isolates of Escherichia coli, Serratia marcescens and Staphylococcus aureus were obtained

from Department of biology/College of Science/Al-Mustansiriyah University/Baghdad / Iraq.

Synthesis of iron oxide nanoparticles by Lactobacillus fermentum

In a typical procedure of nanoparticles synthesis, Lactobacillus fermentum was individually

inoculated into sterilized 250 ml of whole milk in 500 ml flask and incubated for curdling at

37°C for 24 hours. The whey was collected by coarse filtration (Whatman 40).[18]

The filtrate

was pale yellow in appearance, and the pH was typically 4.4. To 5 mL of whey solution taken

in a test tube, 1 mg of Fe2O3 was added and Stirred, then kept in the laboratory under ambient

conditions for 72h. Control was whey without Fe2O3.

Characterization of iron oxide nanoparticles by Atomic Force Microscopy

Atomic Force Microscopy image was taken using Park system AFM XE 100. The aqueous

iron oxide nanoparticles were deposited onto a freshly cleaved mica substrate. The sample

aliquot was left for 1 min and then washed with deionized water and left to dry for15 min.

The images were obtained by scanning the mica in air innon – contact mode.[19]

Antibacterial activity of iron oxide nanoparticles synthesized by Lactobacillus

fermentum

Iron oxide nanoparticles synthesized by Lactobacillus fermentum was screened for their

antibacterial effect against pathogenic bacteria using co - culture technique. The bacterial

culture of pathogenic bacteria was grown in nutrient broth with a ratio (1:1) (iron oxide

nanoparticles solution: nutrient broth), the control medium contained nutrient broth only.[16]

Co-cultures and control were incubated at 37oC for 24 h. After the incubation 1ml of each

cultures were serially diluted upto 10-1

to 10-8

. Then 0.1ml of 10-8

dilution sample was taken

and spreaded on nutrient agar plates. The plates were incubated at37oC for 24 h. The colonies

were counted and the inhibition activity was evaluated after 24 h and calculated percent

reduction of bacteria using the following equation described as Ghosh et al.[20]

:

R(%)=(A−B)/A ×100

R=the reduction rate,A= the number of bacterial colonies from control medium and B= the

number of bacterial colonies from treated with iron oxide nanoparticles.

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Antibiotic susceptibility testing

The Antimicrobial susceptibility of pathogenic isolates was done by using Kirby-Bauer disc

diffusion technique on Mueller Hinton agar [Oxoid, England] using overnight culture at a 0.5

McFarland standard followed by incubation at 35 oC for 16 to 18 h. following Clinical and

Laboratory Standards Institute (CLSI) guidelines[21]

with commercially available

antimicrobial discs (Bioanalyse/Turkey).Isolates were tested against the following

antimicrobial agents: Azithromycin (AZM 15μg), Ceftriaxon (CRO 30μg), Aztreonam

(ATM 30 μg), Ceftazidime (CAZ 30μg), Amoxycillin /clavulanic acid(AMC 30μg), Nalidixic

acid(NA 30μg), Tetracyclin (TE 30μg), Cephalexin (CL 30μg) and Cloxacillin (CX1 1μg).

Evaluation of combined effect between Antibiotics and synthesized iron oxide

nanoparticles

To determine combined effects between antibiotics and synthesized iron oxide nanoparticles,

as described by Roy et al.[22]

with modification,each standard paper disc of antibiotics

(mention above) was further impregnated with iron oxide nanoparticles solution. A single

colony of pathogenic bacterial isolates was grown over night in Muller-Hinton broth medium

at 37Co. The inoculums were prepared by diluting the overnight cultures with 0.9% NaCl to a

0.5 McFarland standard and were applied to the plates along with the standard and prepared

disks containing of iron oxide nanoparticles. After incubation at 37Co for 24 hour, the zones

of inhibition were measured.

Antibiofilm effect of iron oxide nanoparticles

Coating of iron oxide nanoparticles on catheter

The collected catheter was cut in to 1.5cm pieces and sterilized .The cut pieces of the catheter

completely immersed iron oxide nanoparticle suspension and kept in 37ºCfor 24 hrs. Placed

on blotting paper to remove excess suspension and allowed to dry at 50ºc.[23]

Biofilm inhibition assay

The iron oxide nanoparticles coated catheter pieces and control (non coated catheter pieces)

were immersed in 10ml of 24hrs bacterial culture, incubated at 37ºc for 24hrs. After

incubation period all catheter pieces (treated and control catheter) was stained with

0.1%weight by volume of crystal violet solution for 30min at room temperature, after

staining the catheter was washed with 95% of ethanol for 3 times at room temperature, the

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washed solution was collected and read spectrophotometrically at 620nm. The percentage of

biofilm inhibition was calculated as equation described by Namasivayam et al.[23]

% of inhibition = × 100

RESULTS AND DISCUSSION

Synthesis and Characterization of iron oxide nanoparticles

The formation of biosynthesized iron oxide nanoparticles by L. fermentum was observed by

the color changes. Unreduced Fe2O3 was brick red in color whereas Fe3O4 nanoparticles were

dark brown in color, this result agree with.[24]

The particle size of biosynthesized iron oxide nanoparticles was analysed by Atomic Force

Microscopy [AFM] .AFM was used to view the nanoparticles both in surface and three

Dimensional view , and found the average size of particles 68.28 nm Fig. [1,2]. Sundaram et

al.[24]

showed that the biosynthesized iron oxide nanoparticles using Bacillus subtilis were

spherical, with size in the range of (60-80) nm.

Several mechanisms have been proposed to clearify the mechanism of metal oxide

nanoparticles production using microorganisms. The enzymes of the Bacteria play a role in

the bioreduction operation, nanoparticles are biosynthesized when Bacteria capture target

ions from their environment then converted the metal ions in to the elemental metal through

enzymes generated by the cell activities, it can be intracellular and extracellular synthesis

according to the places where nanoparticles are formed.[25]

Lactobacillus have got cell wall

with negative charge which attracts electrostatically positive charged of metal ions, then

enzymes present in cell wall bioreduce metal ions to nanoparticles.[15]

Fig. 1: Diameter percentage of iron oxide nanoparticles synthesized by L.fermentum.

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Jehan et al. World Journal of Pharmaceutical Research

A

B

Fig.2: Atomic Force Microscopy image of iron oxide nanoparticles synthesized by L.

fermentum. )A,B-Surface and three Dimensional view(

Antibacterial activity of Iron oxide nanoparticles against pathogenic bacteria

Iron oxide nanoparticles synthesized by Lactobacillus fermentum isolate showed inhibition

activity against pathogenic bacteria with reduction of growth reached to (46,35,30)% for S.

aureus, E. coli and S. marcescens respectively(Fig.3).The antibacterial activity of iron oxide

nanoparticles might be via oxidative stress generated by ROS.[26,27]

ROS, including

superoxide radicals (O2–), hydroxyl radicals (–OH), hydrogen peroxide (H2O2), and singlet

oxygen (1O2), can cause damage to proteins and DNA in bacteria.[28]

Tran et al.[29]

showed that metal oxide Fe3O4 could be the source that created ROS leading to

the inhibition of S. aureus. Lee et al.[30]

reported that the inactivation of Escherichia coli by

zero-valent iron nanoparticles could be because of the penetration of the small particles (sizes

ranging from 10–80 nm) into E. coli membranes, nano-Fe0could then react with intracellular

oxygen, leading to oxidative stress and eventually causing disruption of the cell membrane.

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Jehan et al. World Journal of Pharmaceutical Research

(Pathogenic bacteria)

Fig 3: Antibacterial activity of Iron oxide nanoparticles against pathogenic bacteria.

Evaluation of combined effect between Antibiotics and synthesized iron oxide

nanoparticles

The effect of iron oxide nanoparticles combined with different antibiotics was investigated

against pathogenic bacteria using disk diffusion method. The diameter of inhibition zones

(mm) around the different antibiotic discs included (Azithromycin, Ceftriaxon, Aztreonam,

Ceftazidime, Amoxycillin /clavulanic acid, Nalidixic acid, Tetracyclin, Cephalexin and

Cloxacillin) with and without iron oxide nanopatrticles against bacterial isolates were

measured. The antibacterial activities of some antibiotics like cephalexin and Nalidixic acid

have been increased in the presence of iron oxide nanoparticle against E.coli isolate while

others didn’t affect (Table 1).

There is great need of agents to kill bacteria and other microorganisms due to the antibiotic

resistance developed by the bacteria.[31]

Combined use of nanoparticle–antibiotic conjugates

towards decreasing resistance to antibiotic observed for specific bacteria and conventional

antibiotics.[32]

Roy et al.[22]

suggested the mechanisms involving the interaction of nanomaterials with

biological molecules and believed that microorganisms carry a negative charge while metal

oxides carry a positive charge, this cause attraction between microorganism and treated

surface leads to oxidizing of microbe and finaly dead. The combination effect of silver

nanoparticles and ampicillin has become more potential compared to the other antibiotics due

reduction

of

growth

%

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Jehan et al. World Journal of Pharmaceutical Research

to the DNA binding action of the AG nanoparticles and the cell wall lysis action of the

ampicillin.[33]

Antibiotic molecules had many active groups such as hydroxyl and amino groups, which

reacts simply with nanoparticles by chelation, causing synergistic effect.[34]

The combination of metal nanoparticles and antibiotics could increase the antibiotics’

efficacy against resistant bacteria. In addition, nanoparticle–antibiotic conjugates lower the

amount of both agents in the dosage, which reduces harmfulness and increases antimicrobial

properties. Additionally, due to this conjugation, the concentrations of antibiotics were

increased at the location of microbe – antibiotic contact and thus accelerate the binding

between antibiotic and microbes.[33]

Table 1: combined effect between Antibiotics and synthesized iron oxide nanoparticles

against pathogenic bacteria

Bacterial

isolates

AZ

M

AZ

M+

N

C

R

O

CR

O+

N

T

E

T

E+

N

C

L

Cl

+N

C

A

Z

CA

Z+

N

A

M

C

AM

C+

N

A

T

M

AT

M

+N

C

X

1

CX

1+

N

N

A

N

A+

N

S.aureus R R R R R R R R R R R R R R R R R R

E.coli - - R R R R R S R R R R S S - - R S

S.marcesc

ens - - R R R R R R R R R R S S - - R R

R=resistant, S=Sensitive, N = iron oxide nanoparticles, Azithromycin (AZM), Ceftriaxon

(CRO), Aztreonam (ATM), Ceftazidime (CAZ), Amoxycillin /clavulanic acid(AMC),

Nalidixic acid(NA), Tetracyclin (TE), Cephalexin (CL) and Cloxacillin (CX1), - (non tested).

Antibiofilm effect of iron oxide nanoparticles synthesized by L. fermentum against

pathogenic bacteria on catheters.

Anti biofilm effect of iron oxide nanoparticles on coated catheters was observed against S.

aureus and E.coli, iron oxide nanoparticles recorded maximum biofilm inhibition 33.97%

against S. aureus, followed by 16.92% occurred against E.coli, While no inhibition on

biofilm formation of S.marcescens was observed(Table 2). The structure of the cell wall play

important role in tolerance or susceptibility of bacteria in the presence of nanoparticles and its

diffusion inside biofilm matrixes by altering surface from hydrophilic to an highly

hydrophobic towards nanoparticles due to change expression of cell wall proteinase.[35]

The

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Jehan et al. World Journal of Pharmaceutical Research

effect of nanoparticles on preformed biofilms through its diffusion inside biofilm matrix

layers using water channales.[36]

Another study on biofilm formation of S.aureus reported that biofilm growth was reduced

at higher concentrations of iron-oxide and gold nanoparticles compared to absence of

nanoparticles.[37]

Taylor and Webster[38]

found the use of super paramagnetic iron oxide

nanoparticles as a multifunctional platform to prevent biofilm formation by S.epidermidis.

Namasivayam et al.[23]

observed reduction in carbohydrates and proteins of biofilm matrix

derived from S.aureus on coated catheter after treated with Ag nanoparticles.

Table 2: Antibiofilm effect of iron oxide nanoparticles synthesized by L.fermentum

against pathogenic bacteria on catheters

Biofilm inhibition (%) Optical density (O.D)

Bacterial isolates Treatment Control

33.97 0.923 1.398 Staphylococcus aureus

16.92 0.918 1.105 Escherichia coli

-13.78 1.246 1.095 Seratia marcescens

Negative results (-%): No inhibition activity

CONCLUSION

The present study demonstrated the synthesis of iron oxide nanoparticles using locally

Lactobacillus fermentum isolate. The synthesized iron oxide nanoparticles had

antibacterial and anti biofilm effect on coated catheters.

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