Surfaces and Interfaces 15 (2019) 70–77

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Surfaces and Interfaces

journal homepage: www.elsevier.com/locate/surfin

Green fabrication of iron oxide nanoparticles using grey mangrove Avicennia T

marina for antibiofilm activity and in vitro toxicity
Vaikundamoorthy Ramalingama,b, Pandurangan Dhinesha, Subramaniam Sundaramahalingamc,
Rajendran Rajarama,
⁎

a
DNA Barcoding and Marine Genomics Lab, Department of Marine Science, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India
b
Department of Animal Science, Chonbuk National University, Jeonju, Republic of Korea
c
Department of Electrical and Electronics Engineering, Mepco Schlenk Engineering College, Sivakasi, Tamil Nadu, India

ARTICLE INFO ABSTRACT

Keywords: The aim of the present study is a biogenic synthesis of iron oxide nanoparticles (FeO-NPs) using grey mangrove
Avicennia marina Avicennia marina to control marine and pathogenic biofilm forming bacteria. Initially, the synthesized nano-
FeO-NPs particles were characterized by UV-visible spectrometer, scanning electron microscope and transmission electron
Characterization microscope for size and shape, Edax analysis for elemental confirmation, zeta potential for stability, X-ray dif-
Antibiofilm activity
fraction for structure and Fourier transform infrared spectroscopy for functional group analysis. Further, the
Toxicity
antibiofilm activity of FeO-NPs were inhibited the initial attachment and biofilm development of primary biofilm
forming bacterial strains Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Almost 72% of
quorum sensing factors of P. aeruginosa were impeded by 200 µg/ml of FeO-NPs, 63% and 46% for S. aureus and
E. coli respectively. The FeO-NPs also decrease the cell surface hydrophobicity of E. coli, P. aeruginosa and S.
aureus from 16% to 9%, 19% to 11% and 15% to 11% respectively. Moreover, the FeO-NPs inhibit the pro-
duction of exopolysaccharide in E. coli, P. aeruginosa and S. aureus from 90% to 69%, 92% to 65% and 86% to
60% respectively. Further, the FeO-NPs also showed less toxicity against human HBL100 cells using the MTT
assay. In conclusion, the biogenic FeO-NPs could be used as a potential antibiofilm agent against marine and
medical pathogenic biofilm forming bacteria.

1. Introduction can act as carrier for pathogenic organisms and cause diseases like otitis
media, otolaryngologic infections, osteomyelitis, bacterial endocarditis,
In the modern technology, nanoparticles are used for numerous cystic fibrosis and non-healing wounds [7]. A conventional path to
biomedical applications where they facilitate laboratory diagnostics inhibit the biofilm formation of bacteria is using the antimicrobial
and therapeutics [1]. More specifically for drug delivery purposes, the agents. But these bacteria are remains in the presence of high con-
use of green synthesized nanoparticles is attracting increasing attention centration of antimicrobial agents due to the existence of poly-
due to their unique capabilities and their negligible side effects not only saccharides, proteins, nucleic acids, phospholipids and humic sub-
in cancer therapy but also in the treatment of other ailments [2]. Re- stances in extracellular polymeric matrix (EPM) that actively involved
cently researchers focusing on magnetic nanoparticles especially iron in stability of biofilm on surface [8]. The higher antimicrobial re-
oxide nanoparticles (FeO-NPs), due to their various applications in- sistance of biofilm bacteria can be a protective wall to EPM, conse-
cluding terabit magnetic storage devices, catalysis, sensors and high- quently the antimicrobial agents acquire to disturb the EPM and de-
sensitivity biomolecular magnetic resonance imaging (MRI) for medical struct the biofilm bacteria [9]. In recent times the scientists are focusing
diagnosis and therapeutics [3]. Apart from this, the antibiofilm activity on destruction of EPM of biofilm bacteria and to prevent the pre-ex-
of FeO-NPs was not clearly studied. isting biofilms completely [10].
The leading problem in medical, food and marine industries is the In modern technology researchers are focusing on the compounds
attachment of bacteria on solid surfaces called “Biofilms”. These bio- with capability of decrease the gene expression that responsible for
films are a serious concern, developed by bacterial communities [4,5] biofilm formation and quorum sensing factor (QSF) [11]. The QSF are
and difficult to remove after provides shelter for bacteria [6]. Biofilms determined by auto inducer molecules like acylated homoserine lactone

⁎
Corresponding author.
E-mail address: [email protected] (R. Rajaram).

https://doi.org/10.1016/j.surfin.2019.01.008
Received 3 November 2018; Received in revised form 11 January 2019; Accepted 24 January 2019
Available online 28 January 2019
2468-0230/ © 2019 Elsevier B.V. All rights reserved.
V. Ramalingam et al. Surfaces and Interfaces 15 (2019) 70–77

(AHL) is the signal molecules produced in out of the cell and attached biofilm was stained using Live/Dead BacLight Viability Kit (BacLight;
with receptor protein that responsible for expression of genes [12]. N- Molecular Probes, Eugene, Oregon, USA), which employs STYO9 and
acetylcysteine (NAC) is a mucolytic agent that effectively decreases the Propidium iodide (STYO9/PI) and subsequently analyzed with confocal
production of EPM and biofilm formation [13]. Cell Surface Hydro- laser scanning microscope (CLSM - Carl Zeiss) [20]. Further, the biofilm
phobicity (CSH) of microorganism was also determining the biofilm cultures were grown on glass surfaces and treated with FeO-NPs
formation and influences the anti-infective potential of bacteria [14]. (100 µg/ml) for 24 h. After treatment, the glass surfaces were washed
The characterization of CSH is depends upon the existence of hydro- with PBS and stained with acridine orange (0.01%). The biofilm for-
phobic proteins in EPM underneath an exterior fibrillar layer and can be mation was observed under CLSM with 488 nm Ar laser and a
precisely determined using a hydrophobicity microsphere assay (HMA) 500–640 nm band pass emission filters
[15]. The characterization of CSH includes the techniques like sessile
liquid drop contact angle measurement, microbial adhesion to hydro- 2.3.3. Effect of FeO-NPs on QSF
carbon (MATH), infrared spectroscopy, X-ray photoelectron spectro- Three biofilm forming bacteria were sub-cultured in M9 minimal
scopy (XPS), electrophoretic mobility, electron microscopy, retention medium by incubating for 24 h at 37 °C and centrifuged at 8000x g for
on chromatographic resins and adhesion to inanimate materials [16]. In 10 min to separate the cell free supernatant. Equal volume of super-
this background, the present study focusing on to find out the efficiency natant and NPs (25–200 µg/ml) were incubated either in combination
of green synthesized iron oxide nanoparticles (FeO-NPs) in disruption or alone in microtiter plate containing biofilm forming culture super-
of EPM of biofilm bacteria and its chemical characterization. Further, natant (density 0.05 OD at 600 nm) and biofilm formation was esti-
the CSH of biofilm bacteria was characterized using MATH assay and mated [21].
inhibition of QSF by FeO-NPs.
2.3.4. Effect of FeO-NPs on cell surface hydrophobicity
2. Materials and methods Hydrophobicity of biofilm bacteria was estimated using microbial
adhesion to hydrocarbons (MATH) assay and adhesion of bacteria on
2.1. Collection and preparation of plant extract glass with hydrophobic hydrocarbons (toluene) was measured [22].
Briefly, 1 ml of biofilm bacterial culture (E. coli – 1.675; P. aeruginosa –
A grey mangrove Avicennia marina leaves were collected from 1.645; S. aureus – 1.623 at OD 600 nm) was taken in test tube and
Muthupet Mangrove forest (Lat 10° 25′ N: Long 79° 39′ E), Southeast 100 µl of toluene with NP (25–200 µg/ml) was added and vortexed for
Coast of India. Collected plant leaves were washed with sea water and 2 min. After incubation for 10 min. at room temperature, the mixture
sterilized with distilled water, dried and powdered using mortar and was allowed for phase separation and the lower aqueous phase was
pestle. 1 g of leaf powder was mixed with 100 ml of water and kept on measured at 530 nm. The percentage of hydrophobicity was calculated
boiling water at 60 °C for 30 min. After cooling, the extracts were fil- using the measurements of before and after vortex, the biofilm bacterial
tered with Whatman No. 1 filter paper and stored at 4 °C [17]. cells with toluene alone in the tube were used as control.

2.2. Biogenic synthesis and characterization of nanoparticles 2.3.5. Effect of FeO-NPs on EPS production
The production of EPS was quantified in control and FeO-NPs
For synthesis of FeO-NPs, 95 ml aqueous solution of 1 mM Ferric treated biofilm forming bacteria by previously described method [23].
chloride (Himedia) was added with 5 ml extract in 250 ml Erlenmeyer The overnight culture of E. coli, P. aeruginosa, and S. aureus was added
flask and incubated in shaker [18]. The reaction mixture was analyzed to 10 ml LB broth treated with and without FeO-NPs extract and in-
using UV-visible spectrometer and the reduction of ferric chloride was cubated for 24 h at 37 °C. After 24 h of incubation the equal volume of
examined by change in color. The synthesized NPs were characterized Trichloroacetic acid (10%) and acetone was added and incubated at
using UV-visible spectrometer (Shimadzu model 1800), FESEM and 4 °C for overnight. The contents were centrifuged again at 10,000 rpm
Edax (ΣIGMA model, CASL ZEISS, German), Zeta potential (Malvern for 10 min and the pellet was weighed.
Zetasizer, Nano-ZS90), XRD pattern (Bruker AXS Microstar) and FTIR
(SAM-AV spectrum RXI). 2.4. Toxicity assessment

2.3. Antibiofilm activity Human normal HBL100 cells was obtained from NCCS (National
Centre for Cell Sciences, Pune, India). These cell lines were grown in
2.3.1. Bacterial culture high glucose DMEM with 50 mM glutamine, supplemented with 10%
Human clinical pathogens such as E. coli (MH701895) and P. aeru- FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. Cells were
ginosa (MH703431) were collected from Government Hospital, maintained in a humidified 5% CO2 incubator at 37 °C. The cells were
Tiruchirappalli, Tamil Nadu, India. The marine bacteria Staphylococcus seeded in 96-well plates at the density of 1 × 105 cells/well in DMEM
aureus (KF554244) was collected from contaminated area of Cuddalore media supplemented with 10% FBS and incubated at 37 °C in 5% CO2
coast. The collected pathogens were cultured in Luria-Bertani broth incubator. Cells were treated with different concentrations (0–200 μg/
(HiMedia) was used and the 24 h old culture was stored as glycerol ml) of FeO-NPs for 24 h at 37 °C. After 24 h of incubation, 20 μl of MTT
stock at −70 °C. The cultures used for each experiment was grown (5 mg/ml) was added and incubated for 4 h, MTT was aspirated and
(OD600 = 1) under aerobic conditions at 37 °C and shaking condition at 200 μl of DMSO was added to dissolve the formazan crystals [24]. The
150 rpm. absorbance was measured at 570 nm using microplate reader (Bio-rad,
USA). The experiments were conducted in triplicates and the significant
2.3.2. Screening of nanoparticles for biofilm inhibition difference was calculated.
The biofilm formation of E. coli, P. aeruginosa and S. aureus
(KF554244) were determined by previously reported method [19]. The 3. Results and discussion
anti-biofilm activity of FeO-NPs, the E. coli, P. aeruginosa and S. aureus
(density 0.05 OD at 600 nm) were grown on glass pieces (1 × 1 cm) 3.1. Biogenic synthesis and characterization of FeO-NPs
placed in 6 well cell culture plates. The 100 µg/ml concentration of
FeO-NPs were effectively inhibiting the biofilm formation in microtiter The bacterial biofilms are familiar in nature and its development
plate were added to 6 well plates and incubated for 36 h at 30 °C. After leads researchers to investigate many pathogenic diseases caused by
removal of non-adherence cells with phosphate buffered saline, the biofilms. These biofilm forming bacteria can damage the medical

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Fig. 1. Represents the UV-visible spectrum of 1 mM ferric chloride and biogenic synthesized FeO-NPs (a), SEM image of FeO-NPs (b), TEM images of FeO-NPs (c,d),
the histogram of FeO-NPs size (e) and Edax analysis of FeO-NPs (f).

devices such as urinary catheters, hemodialysis equipment and medical ∼10 nm [25,26], 15 nm [27] and 64 nm [28] were reported. Moreover,
and dental implants that increases the risk of patient infection. The the Edax analysis showed the presence of Fe element (54%) and the
present study involved in inhibiting the attachment of biofilm forming oxygen molecules (22%) in FeO-NPs (Fig. 1f) and confirmed the green
bacteria on solid surface at initial stage of adherence using FeO-NPs. fabrication of FeO-NPs is highly pure and without any impurities.
The extracts of A. marina were used for reduction of FeCl3 into nano- The XRD pattern of FeO-NPs was showed the presence of FeO at five
materials was confirmed by spectrometrically. The synthesis of NPs was peaks (Fig. 2a) 36.42, 42.24, 61.29, 73.24 and 77.07 were corresponds
characterized by strong surface plasmon peak at 478 nm in UV-visible to (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) planes of FeO (PDF No.:
spectrophotometer (Fig. 1a). The size of FeO-NPs was analyzed using 46–1312) and also the FeO-NP was cubic crystal in nature and con-
FESEM and the results showed that size ranges from 10–25 nm with firmed with JCPDS (Joint Committee on Powder Diffraction Standards).
spherical shape (Fig. 1b). Additionally, the spherical shape of FeO-NPs FTIR spectrum of A. marina and FeO-NPs (Fig. 2b) showed a broad peak
was analyzed with TEM (Fig. 1c & d) and the results were confirmed the at 1383 cm−1, 1635 cm−1 and 3440 cm−1 that corresponds to alco-
size of NPs (10–25 nm) with FESEM and the average size of the FeO-NPs holic OeH stretching band, hydroxyl bending mode and hydroxyl
was found to be 16 nm (Fig. 1e). Previously, the size of the NPs was functional group of alcohol. The presence of γ-Fe2O3 NPs was at five

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Fig. 2. XRD pattern (a), the FTIR spectrum of A. marina and FeO-NPs (b) and the zeta potential of NPs (c).

peaks correspondence to (1 1 1), (2 2 0), (3 1 1), (2 2 2), (4 0 0), (4 2 2),

(5 1 1) and (4 4 0) [29]. The peak from 570–625 cm−1 represents the
presence of FeO stretching bend [30]. The average zeta potential of
FeO-NPs was measured as −17.2 mv (Fig. 2c) showed that the NPs are
stable due to the magnetic property of the FeO-NPs.

3.2. Antibiofilm activity of FeO-NPs using microtiter plate assay

Initially, the biofilm forming efficacy was estimated using 96 well

microtiter plate method and the results showed that E. coli and P. aer-
uginosa were strongly adherent and S. aureus was moderately adherent
on surface of plate. The antibiofilm activity of FeO-NPs was assessed
using concentration dependent method in microtiter plate assay against
E. coli, P. aeruginosa and S. aureus (Fig. 3). The activity of NPs was
commensurate and slightly higher for P. aeruginosa at the concentration
of 200 µg/ml, the biofilm disruption was 84%, 79% and 68% on P.
aeruginosa, E. coli and S. aureus respectively. The present results were
compared with the previous results [19, 31] revealed that nanomater-
ials were effectively involved in biofilm disturbance.
The competence of FeO-NPs in reduction of growth rate of biofilm
Fig. 3. Effect of different concentration of FeO-NPs on biofilm formation
bacteria was measured using growth curve analysis in the presence and
measured using 96 microtiter plate assays. Error bar represents the mean
absence of NPs (25–200 µg/ml). The outcome of growth curve analysis (n = 3).
showed NPs (200 µg/ml) were effectively inhibiting the growth of
biofilm bacteria P. aeruginosa (2.453 ± 0.05 to 1.624 ± 0.03), E. coli
(2.157 ± 0.02 to 1.483 ± 0.03) and S. aureus (2.236 ± 0.06 to 1.581) was dose dependent and the complete inhibition by E. coli and P. aer-
after 24 h of period. The antibiofilm effect of nanomaterials was species uginosa was achieved at 28 and 52 µg/ml of NPs [33]. More than 65% of
specific and it effectively disturbed the biofilm formation at low con- biofilm formation was inhibited at 2 ppm concentration of chitosan-
centration (5 µg/ml) [32]. The antibiofilm activity of nanomaterials stabilized NPs in P. aeruginosa and 5 ppm for S. aureus [34].

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Fig. 4. CLSM shows the effect of FeO-NPs (100 µg/ml) on inhibition of biofilm formation on glass surface.

3.3. CLSM analysis bacteria was observed in the presence of iron-oxide nanoparticles
compared to control [39].
The confocal microscopic analysis also evaluated to determine the
efficacy of NPs against biofilm formation on solid surfaces. This tech- 3.5. Effect of FeO-NPs on QSF
nique proved the inhibition of biofilm formation was due to the in-
creased concentration of NPs, although the low concentration of NPs The ability of NPs to inhibit the QSF was assessed by evaluating the
showed significant inhibition. The complete inhibition of biofilm was competence between NPs and culture supernatant. Roughly 72% of QSF
detected at 100 µg/ml concentration of NPs in P. aeruginosa and mod- inhibiting activity was obtained in P. aeruginosa at 200 µg/ml of NPs
erate activity on biofilm formation was observed in E. coli and S. aureus and 63% for S. aureus and 46% for E. coli (Fig. 6). The different con-
at 100 µg/ml of concentration (Fig. 4). 5 mg/ml concentration of FeO- centration of iron and oxygen affect the quorum sensing regulatory
NPs effectively inhibits the formation of biofilm by E. faecalis, B. subtilis protein lasR. The increase of iron concentration effectively decreases
C. krusei, P. aeruginosa and E. coli [35]. The chemically synthesized FeO- the β—galactosidase activity leads to the expression of lasR that de-
NPs were decreased the number of bacteria at 2 mg/ml concentration creases the virulence activity of bacteria [40]. The probability of po-
[36] (Taylor and Webster, 2009). Further, FeO-NPs also inhibit the tential inhibition of biofilm formation suggested that the biofilm in-
growth of biofilm forming bacteria. After 48h treatment of super- hibiting NPs were interacted with membrane biofilm bacteria and
paramagnetic FeO-NPs (10 µg/ml), 65% of dead S. epidermis bacteria induces the oxidative stress [41].
were quantified by fluorescence microscopic method [36] (Fig. 5).
3.6. Effect of FeO-NPs on hydrophobicity of biofilm bacteria
3.4. Live and dead cells staining assay
CSH plays an important role in initial adhesion of bacteria on solid
Moreover, the live and dead cells were also visualized using SYTO9 surfaces. In our result, the initial hydrophobicity was 16%, 19% and
and propidium iodide staining method. The results indicated that in- 15% for E. coli, P. aeruginosa and S. aureus respectively (Fig. 7). After
crease the concentration of FeO-NPs increase the number of dead cells. 24 h treatment with 200 µg/ml of NPs, the CSH was decreased to 9%,
Among the three bacteria, S. aureus was highly killed followed by P. 11% and 11% for E. coli, P. aeruginosa and S. aureus respectively. The
aeruginosa and E. coli by FeO-NPs. Previously, 24 h treatment with FeO- increase or decrease of absorbance at 530 nm depends upon the pre-
NPs (200 mg/ml) was decrease the P. aeruginosa attachment on glass sence of FeO-NPs on cell wall of biofilm bacteria. FeO-NPs were at-
slide and the complete detachment was observed in NPs combined with tached on the cell wall of biofilm bacteria and increase the interaction
10 mM of fructose metabolites [37]. Nguyen et al. [38] reported that with hydrocarbons that leads to hydrophobicity on cell wall. Recently,
treatments with FeO-NPs induces 19.7% decrease in biofilms which the single dosage of nanomaterials with numerous corrosion rates could
may be due to the presence of iron in the nanoparticles. There is a be adequate to increase the antimicrobial resistance over the MIC level
significant reduction (P < 0.05) in biofilm growth on all the three and evade the biofilm formation [42]. The increase of absorbance was

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Fig. 5. Effect of FeO-NPs on biofilm formation of E. coli, P. aeruginosa and S. aureus using live/dead cells staining assay.

due to the raise of boundary between oil and water during the phase S. aureus respectively (Fig. 8). These findings suggested that the bio-
separation in MATH assay and vice versa for decrease. genic FeO-NPs can act as a potential antibiofilm agent. Recently, Le-
wisoscar et al. [45] reported that the nanoparticles effectively inhibited
3.7. Effect of FeO-NPs on EPS production of biofilm bacteria the EPS production and thus resulted into reduce the EPS efficiency that
prevent the penetration of antimicrobial compounds and provide re-
EPS represents the house of biofilms and determines the life of sistance to antimicrobial agents.
biofilm formation, the biofilm forming ability of microorganisms is lost
by inhibiting the EPS production. Previously many reports stated that 3.8. Toxicity assessment of FeO-NPs
EPS plays a crucial role in the biofilm formation and by destroying the
EPS leads to inhibition of biofilm formation [43,44]. Among the tested Further, the excellent antibiofilm activity of FeO-NPs was assessed
concentration of FeO-NPs, almost 69%, 65% and 60% of EPS produc- for toxicity against human normal cell lines using MTT assay. The re-
tion was inhibited in E. coli, P. aeruginosa and S. aureus respectively by sults showed that the increasing the concentration of FeO-NPs from 0 to
200 µg/ml of NPs after 24 h treatment. Initially, 90%, 92% and 86% of 200 µg/ml are not cause the viability of cells. Instead, the number of
EPS was produced by biofilm forming bacteria E. coli, P. aeruginosa and cells at higher concentration after 24 h treatment with FeO-NPs was

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Fig. 8. Inhibition of EPS production of biofilm forming bacteria using biogenic

Fig. 6. Competence between QSF and different concentrations of FeO-NPs. FeO-NPs. Error bar represents the mean (n = 3).
Error bar represents the mean (n = 3).

Fig. 9. Toxicity assessment of FeO-NPs against HBL100 cells using MTT assay.

Fig. 7. Inhibitory effect of FeO-NPs on the activity of cell surface hydro-

Acknowledgment
phobicity using MATH assay. Error bar represents the mean (n = 3).

The authors gratefully acknowledge the authorities of

82% reveals the biogenic fabricated FeO-NPs has less toxicity in human Bharathidasan University for providing CLSM facility under the DST-
cell lines (Fig. 9). A comparative study between iron oxide nano- PURSE scheme (SR/FT/LS-113/2009).
particles and human normal cell has demonstrated that FeO-NPs not
toxic or they contain the lowest cytotoxicity effect has been reported Conflict of interest
previously [46].
Authors declares that the there is no conflict of interest to disclose.

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