A novel antimicrobial therapy for the control of Aeromonas hydrophila infection in aquaculture using marine polysaccharide coated gold nanoparticle

Accepted Manuscript A novel antimicrobial therapy for the control of Aeromonas hydrophila infection in aquaculture using marine polysaccharide coated gold nanoparticle Sekar Vijayakumar, Baskaralingam Vaseeharan, Balasubramanian Malaikozhundan, Narayanan Gobi, Samuthirapandian Ravichandran, Sellamuthu Karthi, Balasubramaniem Ashok kumar, Natesan Sivakumar PII:

S0882-4010(17)30485-0

DOI:

10.1016/j.micpath.2017.06.029

Reference:

YMPAT 2321

To appear in:

Microbial Pathogenesis

Received Date: 1 May 2017 Revised Date:

8 June 2017

Accepted Date: 21 June 2017

Please cite this article as: Vijayakumar S, Vaseeharan B, Malaikozhundan B, Gobi N, Ravichandran S, Karthi S, Ashok kumar B, Sivakumar N, A novel antimicrobial therapy for the control of Aeromonas hydrophila infection in aquaculture using marine polysaccharide coated gold nanoparticle, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.06.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A novel antimicrobial therapy for the control of Aeromonas hydrophila infection in aquaculture using marine polysaccharide coated gold nanoparticle

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Sekar Vijayakumara, Baskaralingam Vaseeharana*, Balasubramanian Malaikozhundana, Narayanan Gobia, SamuthirapandianRavichandranb, Sellamuthu Karthic, Balasubramaniem Ashok kumarc, Natesan Sivakumard a

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Nanobiosciences and Nanopharmacology Division, Biomaterials and Biotechnology in Animal

Health Lab, Department of Animal Health and Management, Science campus 6th floor, Alagappa

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University, Karaikudi 630004, Tamil Nadu, India.

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b

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Tamil Nadu, India.

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c

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Madurai - 625 021, Tamil Nadu, India.

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d

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University, Madurai - 625 021, Tamil Nadu, India.

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.

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-------------------------------------------------------------------------------------------------------------*Corresponding author

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Dr.B. Vaseeharan,

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Professor & Head

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Nanobiosciences and Nanopharmacology Division, Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management,

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Science campus 6th floor

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Alagappa University, Karaikudi- 630 004, Tamil Nadu, India.

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Tel: + 91 4565 225682.

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Fax: + 91 4565 225202.

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E.mail: [email protected], [email protected]

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Center of Advanced Study in Marine Biology, Annamalai University, Parangipettai- 608 502,

Department of Genetic Engineering , School of Biotechnology, Madurai Kamaraj University,

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Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj

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Abstract

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In the present study, we prepared fucoidan coated Au-NPs, known as gold nanoparticles of

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fucoidan (Fu-AuNPs), and examined its effect on the antimicrobial activity against Aeromonas

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hydrophila. The green synthesized Fu-AuNPs were bio-physically characterized by Ultraviolet–

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visible (UV-Vis) spectroscopy, X-ray Diffraction (XRD), Fourier Transform Infrared

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spectroscopy (FTIR), Higher Transmission Electron Microscopy (HR-TEM), Zeta potential

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analysis and Energy Dispersive X-ray spectroscopy (EDX). Fu-AuNPs were crystalline in nature,

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spherical to triangular in shape, with particle size ranging within 10-100 nm. The synthesized Fu-

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AuNPs at 100 µg mL-1 showed inhibition zone against A. hydrophila 23.2 mm which is much

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higher (17.3 mm) than that of commercial antibiotic chloramphenicol The biofilm inhibition

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activity of Fu-AuNPs against Gram negative (Aeromonas hydrophila) was higher. Light and

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confocal laser scanning microscopic observations showed that the Fu-AuNPs at 100 µg mL-1

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inhibited

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AuNPs were effective in inhibiting the viability of human cervical cancer cells (He La)

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at 100 µg mL-1. In another experiment the antibacterial effect of Fu-AuNPs on tilapia

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Oreochromis mossambicus were evaluated in vivo. The mortality rate of O. mossambicus

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that infected by A. hydrophila was much higher (90%), whereas the mortality of O. mossambicus

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that received Fu-AuNPs and then challenged with A. hydrophia reduced to 30%. The Fu-AuNPs

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had antibacterial activity against Gram negative bacteria, Aeromonas hydrophila.

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Keywords: fucoidan; goldnanoparticles; TEM; Aeromonas hydrophila; antibiofilm; anticancer

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of

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biofilm

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cytotoxicity

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1. Introduction

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World aquaculture is a fastest-growing animal food source for increasing fish supply with an

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average increase of 6% per year, and it has made a great contribution to the production of

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protein-rich food for human consumption [1,2]. However, aquaculture practices have

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encountered serious issues like disease outbreaks caused by microbial pathogens [3]. Aeromonas

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an important pathogen in many aquatic animal species or food [4]. Aeromonas hydrophila causes

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skin ulceration, tail or fin rot, and fatal hemorrhagic septicemia in fish [5]. It and is known to

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infect common carp, gold-fish (Carassius auratus), and silver catfish (Rhamdia quelen), leading

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to huge economic losses in aquaculture [5,6]. Fish farmers use antibiotics to control Aeromonas

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infections. However, use of antibiotics may pollute environment and in turn affects human health

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[7]. Long-term use of antibiotics leads to drug resistance of pathogens and reduces the

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effectiveness of the antibiotics. Tilapia is hardy individuals that are easy to harvest, making them

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a good aquaculture species. The culture of O. mossambicus in freshwater aquaculture has

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suffered due to bacterial infections, which results in heavy losses and causes economic loss to

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fish farmers [8]. Development of, an effective alternative way in replace the use of antimicrobial

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agents is of primary concern. Marine polymers are interesting biomaterials and, can be used for

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obtaining biomimetic nanoparticles with tunable surface properties. Among the marine derived

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polysaccharides, fucoidan

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marine brown seaweeds, Fucus vesiculosus it contains large proportions of L-fucose and sulfate.

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[9]. Fucoidan possess antibacterial, antiviral, antitumor, and anticoagulant activities. [9,10 ,11].

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They have been developed as a specialised type of nutraceutical and food supplement [10].

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Fucoidan was used as an immuno-therapeutic polymer and is an excellent drug candidate for

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pharmaceutical applications [11]. In vivo studies indicate that fish fed diets containing fucoidan

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is a naturally occurring sulfated polysaccharide extracted from

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exhibit enhanced growth and resistance against pathogen [12]. However, none is known on the

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effect of fucoidan coated nanoparticles in the resistance against pathogen.

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In aquaculture, nanotechnology has been used the improvement of the quality of ingredients in

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food formulations, antifouling coatings, antibacterials for tanks and packaging of sea food

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products, and environmental remediation systems [13]. Nanoparticles exhibit specific

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characteristics such as size, distribution and morphology. Metal nanoparticles are new generation

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nanomaterials with biomedical and therapeutic applications. Among these, gold nanoparticles

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(AuNPs) owing to their desirable optical, electrical, and chemical properties have received

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attention recently [14]. The antibacterial, antibiofilm and cytotoxic effects of Nigella sativa

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essential oil coated AuNPs on human A549 lung cancer cells have been studied [15]. In the

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present study, the antibacterial and antibiofilm effects of fucoidan coated AuNPs were tested

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against A. hydrophila. In addition, the protective effect of Fu-AuNPs on the freshwater tilapia

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infected with A. hydrophila were examined in vivo. Furthermore, the anticancer attributes of

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fucoidan coated AuNPs (Fu-AuNPs) was investigated on human cervical cancer cells.

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cervical cancer, a malignant growth of cells in the area of cervix, is the fourth most common

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cancer in women worldwide. It was estimated that 528,000 new cases of cervical cancer and

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266,000 deaths occurred in 2012. In the present study, the anticancer effect of fucoidan coated

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AuNPs

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2. Materials and methods

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2.1. Chemicals used

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Chloroauric acid (HAuCl4) (254169), Fucoidan from Fucus vesiculosus, (F8190 ≥95%) ,

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whatman filter paper (WHA10348903), Phosphate buffered saline (PBS) tablets (pH 7.4),

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was

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cancer

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Dulbecco Modified Eagle Medium (DMEM), Fetal bovine serum (FBS), Cell counting kit-8

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(CCK-8), Fluorescent dye propidium iodide (PI) (33342), acridine orange (235474) and crystal

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violet (c3886) were purchased from Sigma Aldrich, Mumbai, India. Nutrient broth (NB), Luria

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Bertani agar (LBA) and Luria Bertani broth (LBB) were purchased from Hi Media, Mumbai,

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India. Mueller Hinton Agar (MHA) obtained from Hi Media, Mumbai, India. Gram negative

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Aeromonas hydrophila (ATCC: 7966) were commercially purchased from American type culture

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collection (ATCC), Manassas, Virginia (USA). Ultra-pure deionized water from PURITE (18

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MΩ, UK) system was used. The chemicals used were of analytical grade. The glass wares

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(Borosil, Mumbai, India) used for experimental purposes were properly washed, sanitized and

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autoclaved.

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2.2. Green synthesis and characterization of Fu-AuNPs

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Chloroauric acid (HAuCl4) was used for AuNPs synthesis. Briefly, a stock solution of fucoidan

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was prepared by dissolving 1g in 100ml of distilled water to get a concentration of 0.01g/ml.

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About 2 ml of fucoidan stock solutions was mixed with 25 ml of 1 Mm HAuCl4 and kept at 353

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K for 20 min. The characteristic change in color from milky white to dark ruby red indicated the

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synthesis of gold nanoparticles [16]. The concentration of fucoidan per ml of Fu-AuNPs is 0.08g/

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8mg/ 8000 µg. The synthesized Fu-AuNPs were diluted to get a desired concentration of 25, 50

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and 100 µg mL-1 for the following studies.

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Fu-AuNPs were physico-chemically characterized by UV–Visible spectroscopy, a very

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useful technique for the analysis of nanoparticles. UV-Vis spectra were recorded using a

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Shimadzu UV- 1800pc spectrophotometer at wavelengths ranging between 200 and 800 nm. The

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crystalline nature of the synthesized Fu-AuNPs was determined by XRD analysis using X-Ray 5

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diffractometer (X’Pert-PRO). The high resolution on XRD patterns measured at 3 KW with Cu

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target using a scintillation counter (k = 1.54 A°) at 40 kV and 40 mA was recorded in the range

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of 2h = 10 –80θ.

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The possible functional groups and changes in the surface chemical bondings were characterized

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using Fourier Transform Infrared (FTIR) spectroscopy (Nicolet Avatar series 330) ranging from

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500 to 4000 cm-1. Fu-AuNPs samples were uniformly mixed with potassium bromide (KBr) and

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compressed with a hydraulic press to prepare disks, which were then used for FTIR analysis.

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The surface charge of the particle was measured by zeta potential using a Zeta sizer Nano

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ZS90 (Malvern Instruments, UK). The particle size and morphology of the Fu-AuNPs was

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observed under high-resolution transmission electron microscopy (HR-TEM, Tecnai 12, Philips,

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120 kV). The sample was prepared by placing a drop of the AuNPs solution on a carbon coated

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copper grid (300 meshes), followed by drying at room temperature (20 ºC) for 30 min. The

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elemental composition of Fu-AuNPs was determined by EDX spectroscopy (IncaEnergy-350,

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Oxford Co., UK).

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2.3. In vitro antibacterial activity of Fu-AuNPs

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The agar well diffusion method [17] was used to screen the antibacterial activity of Fu-AuNPs

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against the fish specific pathogenic Gram negative Aeromonas hydrophila (ATCC7966) bacteria.

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Briefly, the MHA plates were prepared by pouring 15 ml of molten media into sterile petri

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plates. The plates were allowed to solidify for 5 min and 0.1% inoculum suspension of bacterial

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strains was swabbed uniformly and the inoculum was allowed to dry for 5 min. Then, wells were

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made on the plate using well puncher for loading the Fu-AuNPs. 50 µl of different concentration

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of Fu-AuNPs (25, 50, and 100 µg mL-1)) was loaded on to the wells. The compound was allowed

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to diffuse for 5 min and the plates were incubated at 37 °C for 24 h. after incubation, the

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inhibition zones formed around the wells were measured with transparent ruler in millimeter.

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The antibacterial efficacy of Fu-AuNPs was compared with positive control commercial

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antibiotic chloramphenicol, bare chloroauric acid (HAuCl4) and fucoidan crude extracts

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respectively.

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2.4. Minimum Inhibitory Concentration (MIC) of Fu-Au NPs

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The Minimum Inhibitory Concentration (MIC) of bare chloroauric acid (HAuCl4), fucoidan

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crude extracts and Fu-AuNPs were determined by the method of Burt [16]. Tubes with 5ml of

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Luria-Bertani (LB) broth containing various concentrations of bare chloroauric acid (HAuCl4),

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fucoidan crude extracts and Fu-AuNPs ranging from 5 to 9.5µg mL-1 were inoculated with

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200µl of 106 CFU mL-1 of standardized suspensions of bacterial culture. The tubes were

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incubated in orbital shaker (180 rpm) for 24 h at 37 °C. About 100µl from each dilution tube was

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plated in MHA plates and incubated for overnight at 37 °C. The results were recorded by

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comparing plates with bare chloroauric acid (HAuCl4) and fucoidan crude extracts and positive

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control (i.e. chloramphenicol).

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2.5. Antibiofilm assay

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To analyze the ability of Fu - AuNPs to prevent the biofilm formation of Aeromonas

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hydrophila (ATCC:7966), bacterial colonies (1 x 106 CFU mL-1) were allowed to grow on glass

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pieces (diameter 1x1cm) placed in 24-well polystyrene plates containing 1ml of nutrient broth

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supplemented with different concentrations of Fu-AuNPs (25, 50 and 100 µg mL-1). The plates

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were then incubated for 24h at 37°C. Simultaneously, a control was set up by growing the

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bacterial colonies (1 x 106 CFU ml-1) in glass pieces placed in 24-well polystyrene plates loaded

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with different concentrations of bare chloroauric acid (HAuCl4) and fucoidan crude extracts (25,

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50 and 100 µg ml-1). After incubation, the glass pieces were stained with 0.04% crystal violet and

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visualized under a inverted research microscope (ECLIPSE Ti100) at 40x magnification.

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Similarly, glass pieces with biofilms grown as above were washed with PBS, stained with 0.1%

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acridine orange and the biofilm growth was quantified under a confocal laser scanning

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microscope (CLSM- Carl Zeiss LSM 710, Carl Zeiss, Germany). The Z-stack analysis (surface

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topography and three-dimensional architecture) was done with the Zen 2009 software (Carl

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Zeiss, Germany). To measure the biofilm thickness, sections were scanned and Z-stacks were

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acquired at z step-size of 0.388 µm. Each field size was 455 µm by 455 µm at 20× magnification.

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Microscope images were acquired with the Zen 2009 image software.

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2.6. BATH assay

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Bacterial adhesion to hydrocarbons (BATH) (hydrophobicity index) assay were performed

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following the methods of Zhang and Miller [18]. Briefly, cells from the overnight culture of A.

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hydrophila (control and Fu-AuNPs treated) were resuspended in MHB and adjusted to an OD at

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595 nm of 1.0 ± 0.01. Toluene (1 mL) was added to the cell suspension and was vortexed for 1

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min. The mixture was then allowed to settle and separate for 30 min before the OD of the

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aqueous phase was measured. The hydrophobicity index (HI) of bacterial cells was calculated

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using the formula below

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[(A0 − A) A0 −1] × 100

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where, A0 and A are the initial and final optical densities of the aqueous phase respectively. The

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results were expressed as the proportion of cells excluded from the aqueous phase, as determined

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using above equation [19].

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2.7. In vivo studies

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2.7.1. Experimental animal and their maintenance

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Tilapia, Oreochromis mossambicus was obtained from aquaculture farms Kallupatti village,

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Tamil Nadu,India. The initial body length and weight of the stock fish (n =80) were 14.6± 0.64

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cm and 53.3 ± 8.35g respectively. Fish were transported to the laboratory in large plastic tanks

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with continuous aeration. Fish were kept for 2 weeks in a 50 L-1 glass tank supplied with

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unceasingly aerated and dechlorinated tap water. Water temperature (25± °C) dissolved oxygen

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(6.5–7.8 mg L-1) and pH (7.1–7.3) were maintained. During the acclimatization period, fish were

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fed twice a day with commercial feed pellets (Al-Manzala factory at El-Dakahlia province) (20

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% crude protein, 4 % crude fat, 5 % crude fiber, 12 % crude ash, and 10 % crude moisture) with

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12/12-h light/dark photoperiod. Fish behaviors were observed, and any fish with unusual

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performances were excluded. To avoid any contamination by the fecal materials of fish, the

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water was renewed daily and dead fish were removed [20].

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2.7.2. In vivo antibacterial studies

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The experiment consists of 3 groups and 10 fish per group was maintained. The experiment was

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performed for 72h and triplicates were maintained.

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Group 1: Control (50µl of physiological saline)

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Group 2: infected via intraperitoneally with 50 µl (6.0×108 CFU/ mL-1) of A. hydrophila

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Group 3: infected via intraperitoneally with 50 µl (6.0×108 CFU/ mL-1) of A. hydrophila+100µl

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of Fu-AuNPs.

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During the experiment period, temperature (25± °C) dissolved oxygen (6.5–7.8 mg L-1) and pH

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(7.1–7.3) were maintained. Clinical signs, postmortem lesions and mortalities were recorded at

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regular intervals. Dead fish were removed from the aquarium daily [21].

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2.8. Cytotoxicity on human cervical cancer cell (He La cells)

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The inhibitory concentration (IC50) value was calculated using the MTT [3-(4, 5-

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dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. Briefly, human He La cervical

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cancer cell line was procured from National Centre for Cell Science (NCCS), Pune, India. They

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were grown in DMEM (Dulbecco's modified eagle medium) enhanced with 2 mM L-glutamine,

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100 U/ml penicillin, 100 mg/ml streptomycin and 10% FBS. The cells were cultured using 75

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cm2 cell culture flasks at 37°C in a CO2 incubator (95% air, 5% CO2 and 100% relative

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humidity). They were then introduced into 96 well plates (5000 cells in each well) and incubated

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for 24 h. He La (cervical cancer) cells were treated with different concentrations of Fu-AuNPs

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(25, 50, 75 and 100 µg mL-1). Similarly, bare chloroauric acid (HAuCl4) and fucoidan crude

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extract was used as positive control separately (25, 50, 75, and 100 µg mL-1). A respective

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negative control (100 µg ml-1) was prepared using DMEM medium and saline. Following

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treatment, the plates were incubated for 24 h. MTT at 5 mg mL-1 was added to each well and

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incubated for 4 h. Purple color formazone crystals formed were then dissolved in 100 mL of

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dimethyl sulfoxide (DMSO). Optical density was read at 570 nm using ELISA plate reader. The

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percentage of cell viability was calculated by using the following formula:

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OD value of experimental samples

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Percentage of cell viability =

____________________________ × 100 OD value of experimental controls

He La cells were plated into a six well chamber plate at 3 x 105 cells/well. At > 90% confluence,

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the cells were treated with fucoidan crude extract (25 to 100 µg mL-1) for 24 h. The cells were

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washed with PBS fixed in methanol: acetic acid (3:1 v/v) for 10 min. The morphological

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variations were examined under phase contrast microscope (Olympus, Japan) and confocal laser

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scanning microscope (CLSM- Carl Zeiss LSM 710) using a 488nm argon laser and band path

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500-640 band pass emission filter and running Zen 2009 software (Carl Zeiss, Germany).

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2.9. Statistical analysis Experiments were carried out in a randomized block design with three replications. The

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data were analysed using one way analysis of variance (ANOVA) followed by Tukey’s HSD test

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(P<0.05).

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3.0. Results

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3.1. Synthesis and characterization of gold nanoparticles

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After reaction with Au ions, the change in color from milky white to ruby red indicated

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the formation of gold nanoparticles. The synthesis of gold nanoparticles was characterized by

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UV–Vis spectroscopy. The surface plasmon resonance (SPR) bands of Fu- AuNPs were centered

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at 537 nm (Fig.1). XRD pattern of Fu-AuNPs showed four diffraction peaks at 2θ corresponding

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to 38.2 º, 44.4 º, 64.6 º, and 77.7 º that reflected the (111), (200), (220), and (311) planes of the

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face-centered cubic (fcc) crystal structure respectively (Fig.2). In FTIR, the intense absorption

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peak at 3437cm-1 corresponds to the O–H stretching vibrations of phenols and carboxylic acids.

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(Fig.3). A major peak observed at 2921cm−1 represents the C–H stretching vibrations of methyl,

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methylene and methoxy groups. The peak located at 1661cm−1 corresponded to the C=O

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stretching of carboxyl or C=N bending in the amide group. The band observed at 1383cm−1 was

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assigned to C–N stretching or the O–H bending and its shift to 1038cm−1 implicated the role of

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these groups in the interaction with chloroauric acid. The peak at 612 cm-1corresponds to C– H

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stretching of aromatic compounds. The zeta potential of AuNPs synthesized from fucoidan crude

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extracts revealed the negative surface charge of nanoparticles (-23.5 mV) (Fig.4). HR-TEM

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revealed that the gold nanoparticles synthesized from the fucoidan crude extracts exhibited

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spherical to triangular shape. The size of Fu-AuNPs was between 10 to100 nm (Fig.5). The EDX

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spectrum of Fu-AuNPs (Fig.6) revealed a strong and typical absorption peak at 200 keV, which

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could be due to the SPR of the metallic Au nanocrystals, and confirms the synthesis of AuNPs in

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the reaction medium. In addition, the presence of Cu, Cl, C, and O elements were also recorded

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in the EDX spectra.

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3.2. In vitro Antibacterial activity of Fu-AuNPs

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The minimum inhibitory concentration (MIC) of Fu-Au NPs was comparatively lesser

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than that of bare chloroauric acid (HAuCl4) and fucoidan crude extract. The MIC of Fu-Au NPs

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against A. hydrophila was 1.875 µg mL-1. (Fig.7). Fu-Au NPs exhibited greater activity against

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tested bacteria compared to bare chloroauric acid (HAuCl4) and fucoidan crude extracts.

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(Fig.8A). The zone of inhibition against A. hydrophila was 23.2 mm at 100 µg mL-1of Fu-Au

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NPs. (Fig.8 B) On the other hand, chloramphenicol (commercial antibiotic) showed 17.3 mm at

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100 µg mL-1 of inhibition zones against A. hydrophila.

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3.3. Antibiofilm assays

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The light and confocal laser scanning microscopic observation showed well developed

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biofilm formation of A. hydrophila, whereas, treatment with Fu-AuNPs (25, 50 and 100 µg mL-1)

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a significant reduction in biofilm formation was observed in a dose dependent manner (Fig.9 A

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and 9 B). At higher concentration (100 µg mL-1) of Fu-AuNPs, a complete reduction in biofilm

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growth was observed. The thickness of biofilm was reduced (7µm) following treatment with 100

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µg mL-1 Fu -AuNPs compared to control (30µm) (Fig.10 a and b). However, fucoidan crude

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extract inhibited the biofilm of A. hydrophila at only at 300 µg mL-1 (data not shown). On the

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other hand, bare chloroauric acid (HAuCl4) does not show any activity on the biofilm of A.

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hydrophila at tested concentration (25, 50 and 100 µg mL-1) (data not shown).

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3.4. BATH assay Bacteria in the control and bare HAuCl4 treated groups showed higher percentage of

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hydrophobicity i.e. (100% and 85 % respectively). Interestingly, a significant reduction in the

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hydrophobicity was observed (40 %) at 300 µg mL-1 of fucoidan crude extract. However, the

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percentage of hydrophobicity index was significantly reduced after treatment with Fu-AuNPs at

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100 µg mL-1 (90%). (Fig.11).

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3.5. Cytotoxicity on human cervical cancer cell (He La cells)

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The in vitro cytotoxic effects of Fu-AuNPs was evaluated against human cervical cancer cells at

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different concentrations (25, 50, 75 and 100µg mL-1) in comparison with bare HAuCl4 and

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fucoidan crude extract. The viability of cervical cancer cell was decreased when the

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concentration of Fu-AuNPs was increased to 100µg mL-1 (Fig.12A). This attributed that Fu-

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AuNPs are effective in inhibiting the viability of cervical cancer cells. To further confirm the

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cytotoxic effects of Fu-AuNPs on the apoptotic cell morphology, propidium iodide stained cells

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were visualized under phase contrast microscope (Olympus, Japan) and confocal laser scanning

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microscope (CLSM- Carl Zeiss LSM 710) using a 488nm argon laser and band path 500-640

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band pass emission filter and running Zen 2009 software (Carl Zeiss, Germany).

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When compared to PBS saline control, the cervical cancer cells treated with Fu-AuNPs at 100µg

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mL-1. showed nuclear morphological changes such as cell clumping. A noticeable changes in the

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morphology of the cells such as rounding, shrinking and granulation in the cytoplasm and loss of

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membrane stability was observed at 100µg mL-1 after 48 h (Fig.12B a and b) The cytotoxicity

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studies revealed that Fu-AuNPs were more effective in controlling the growth of cervical cancer

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cells compared to PBS saline control. However, fucoidan crude extract at 300µg mL-1

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significantly reduced the cell viability (45 %) and caused least morphological changes compared

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to control. On the other hand, a least cytotoxic (10 %) effects were observed on cervical cancer

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cell treated with bare HAuCl4 at 100µg mL-1.

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3.6. In vivo antibacterial activity

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3.6.1. Examination of pathogenicity of A. hydrophila

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A. hydrophila was found to be highly virulent and caused 90 % mortality of O. mossambicus

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after 48 h post infection. Following infection, O. mossambicus showed clinical signs in the form

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of ulceration of skin, hemorrhage. erythema on the external body surface such as mouth and

311

operculum at pectoral fin followed by fin rot at caudal fin. (data not shown).

312

3.6.2. Protective effects of Fu -AuNPs on the survival of O. mossambicus infected with A.

313

hydrophila

314

The results revealed that the mortality of O. mossambicus infected with A. hydrophila (Group2)

315

was 90% after 72h. Interstingly, significant reduction in the mortality of O. mossambicus was

316

observed (30 %) following challenge with Fu-AuNPs (Group3) (Fig.13).

317

4. Discussion

318

This study reports for the first time the synthesis of fucoidan coated gold nanoparticles by an

319

inexpensive, ecofriendly and rapid method. it was reported that the ruby red color of the gold

320

solution usually indicates the presence of AuNPs, which corresponds to the SPR band around

321

520-530 nm [22]. Normally, the AuNPs having size of less than 25 nm show the SPR band of

322

lower than 530 nm or so-called the intrinsic size region where the wavelength shift in the

323

absorption maxima is not significant and the measured absorbance directly determines the

324

concentration of nanoparticles present [23]. The UV-Vis absorbance spectrum recorded in the

325

present study showed a well defined surface plasmon band centered at 537 nm. which is the

326

characteristic absorbance of gold nanoparticles [22]. The present finding corroborates with the

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results by Vijayakumar et al. [24] and Heath [25] who reported that SPR shift increases with

328

increasing concentration of the plant extract.

329

XRD helps to understand the crystalline nature of nanomaterials. In the present study, XRD

330

diffraction pattern of the AuNPs showed ,four distinctive diffraction peaks at 2θ¼ 38.2, 44.4,

331

64.6, and 77.8 which were assigned to (111), (200) ,(220), and (311) planes of respectively. The

332

peak at 2θ¼ 38.2 was found to be more intense than those of other peaks, which might be due the

333

predominant orientation of (111) plane. Manju et al. [15] and Vijayakuamar et al. [24] observed

334

similar diffraction peaks with gold nanoparticles using Nigella sativa essential oil and Musa

335

paradisica peel extract respectively.

336

Fourier transform infrared spectroscopy (FTIR) was used to identify the possible biomolecules

337

present in fucoidan crude extract which are responsible for capping and efficient stabilization of

338

AuNPs. In the present study, the intense broad absorption peak at 3437 cm-1 represents the O=H

339

stretching vibrations of phenols and carboxylic acids. The shift at 3437 cm-1 indicated the

340

involvement of O=H functional group in the synthesis of nanoparticles. A major peak at 2921

341

cm-1 corresponds to the C=H stretching vibrations of methyl, methylene and methoxy groups.

342

The peak located at 1661 cm-1 was assigned to the C=O stretching in carboxyl or C=N bending in

343

the amide group. The band observed at 1383 cm-1 was assigned to C=N stretching or the O=H

344

bending and its shift to 1034 cm-1 implicated the role of these groups in the interaction with

345

chloroauric acid. The peak at 612 cm-1 corresponds to C=H stretching of aromatic compounds.

346

These results are in agreement with the observations of Manivasagan et.al [26] who reported that

347

a major peak observed at 2930 cm-1 could be assigned to the C=H stretching vibrations. and the

348

peak located at around 2353 cm-1 was attributed to the N=H stretching vibrations or the C=O

349

stretching vibrations.

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Higher resolution Transmission electron microscopy (HR-TEM) is frequently used to determine

351

the morphology, size and shape of nanoparticles. HR-TEM showed that AuNPs formed were

352

mostly spherical in shape with diameter ranges from 10–100 nm. As evidenced by TEM images,

353

individual nanoparticles were uniformly distributed and well stabilized by biopolymer

354

fucoidan.These results are in good agreement with those reported by Vijayakumar et.al [24] for

355

gold nanoparticles synthesized from Musaparadisica peel extract triangle to spherical shape with

356

size between 50nm. Pucci et al. [27] also found that TEM images of PVA-stabilized AuNPs

357

showed spherical shape of nanoparticles with average size of 3–20 nm.The presence of elemental

358

gold in the formed nanoparticles was examined by energy dispersive X-ray (EDX) analysis. The

359

strongest signal appeared at gold region (_2 keV, 38%) confirms the presence of elemental gold

360

in the solution. Due to the strong surface plasmonic effect, the metallic AuNPs are known to

361

exhibit gold signals at _2 keV [26]. In addition, various other peaks were observed presumably

362

due to the presence of other atoms came from fucoidan and grid for sample holding for the TEM

363

analysis.

364

Zeta potential provides information on the surface charge and stability of bio synthesized Fu-

365

AuNPs. The zeta potential of Fu-AuNPs in the present study showed that the particle was

366

negatively charged (−23.5 mV). Zeta potential analysis indicated that capping molecules present

367

on the surface of AuNPs are mainly comprised of negatively charged groups and are the

368

moderately, stable. Our results corroborates with the observations of Manivasagan et al [26] who

369

reported that the zeta potential of gold nanoparticles synthesized using the Doxorubicin-loaded

370

fucoidan capped gold nanoparticles.

371

Gold nanoparticles excerted the antibacterial effect in two ways, they changed the membrane

372

potential and reduced adenosine triphosphate (ATP) synthesis activities, thus reducing the

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metabolism process. Secondly, they declined the subunit of the ribosome for tRNA binding, thus

374

collapsing its biological mechanism. At the same time, they proved to be less toxic to mammal

375

cells [28]. Gold NPs with a small size and enhanced surface area produce some electronic effects

376

which are beneficial for enhancing the surface reactivity of NPs. In addition, the high surface

377

area directly interacted with the microorganism to an enormous extent and hence provided an

378

improved contact with the bacteria. The gold NPs binded to thiol groups of enzymes such as

379

nicotinamide adenine denucleotide (NADH) dehydrogenases and disrupted their respiratory

380

chains with the release of oxygen species, producing oxidative stress. As a result, significant

381

damage occurred in the cell structures and finally led to cell death [29].

382

It was demonstrated that the catalytic and antibacterial mechanism of the gold NPs increases

383

with a decrease in size [30]. In the present study, Fu-AuNPs produced the 20.3 mm inhibition

384

zone against Gram negative A.hydrophila.

385

activity of NsEO-AuNPs was greater against Gram positive S. aureus MTCC 9542 (16 mm) than

386

Gram negative V. harveyi MTCC 7771 (5 mm) at the concentration of 10 µg mL-1. In recent

387

years, biofilm mode of bacterial and fungal growth has posed several problems. According to

388

public announcement from national institute of health, more than 60% of all microbial infection

389

is caused by biofilms [31]. Infections resulting from microbial biofilm formation remain a

390

serious threat to patients worldwide. In order to kill or remove biofilms, anti-microbials must

391

penetrate the polysaccharide matrix to gain access to the microbial cells. Nanotechnology may

392

provide the answer to penetrate such biofilms and reduce biofilm formation by the use of 'nano

393

functionalisation' surface techniques to prevent the biofilm formation.

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Manju et al. [15] reported that the antibacterial

394

Impeding the bacterial adhesion at an early stage can significantly decrease the threat of

395

further biofilm development. In the present study, Fu-AuNPs prominently inhibited A.

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biofilm at 100 µg mL-1. In addition, the light and confocal laser scanning

hydrophila

397

microscopic (CLSM) observation confirmed the reduction in the biofilm architecture of A.

398

hydrophila biofilms treated with 100 µg mL-1 of Fu-AuNPs. This results was consistent with the

399

findings of Choi et al. [32] who reported that interactions of nanosilver with biofilm-forming

400

cells resulted in significant inhibition.

401

The results of BATH assay showed that Fu-AuNPs reduced the hydrophobicity and

402

exopolysaccharide production of A. hydrophila biofilms. The synergistic interactions between

403

fucoidan crude extracts and the gold nanoparticles may inhibited the quorum-sensing molecules,

404

which further leads to the inhibition of biofilm formation. Yan et al. [33] reported that the strong

405

antibacterial activity against MDR bacteria was observed for gold NPs due to their multiple

406

targets and inherent elemental properties. EPS and cell surface hydrophobicity play an important

407

role in bacterium host cell interactions and biofilm architecture [34, 35]. Generally, targeting the

408

hydrophobicity index is a novel way of inhibiting the biofilm formation. The light and confocal

409

laser scanning microscopic observation in the present study, showed well-developed biofilm

410

formation of A. hydrophila.However, after treatment with Fu-AuNPs, a significant reduction in

411

bacterial biofilm was observed in a dose dependent manner. At higher concentration of Fu-

412

AuNPs (100 µg mL-1), A. hydrophila showed disintegrated and recalcitrant biofilm architecture.

413

The percentage of hydrophobicity index also decreased after treatment with Fu-AuNPs such that

414

74% hydrophobicity observed as compared to untreated bacteria. Our results corroborate with

415

the findings of Manju et al. [15] who reported that the hydrophobicity inhibition against

416

Pseudomonas aeruginosa and S. aureus by NsEO-AuNPs was 78% and 46% respectively.

417

Malaikozhundan et al [36] reported that Mc-AgNPs was found to significantly inhibit the biofilm

418

formation of E. faecalis and A. hydrophila at 100 µg mL-1 .The antibiofilm activity of AuNPs

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could be due to inhibition of exopolysaccharide synthesis because metallic nanoparticles impair

420

exopolysaccharide synthesis, which prevents biofilm formation [37].

421

The cytotoxicity of Fu-AuNPs was evaluated against human cervical cancer (He La cells) at

422

different concentrations (25 to 100 µg mL-1). A significant decrease in the cell viability of human

423

cervical cancer (He La cells) was noticed when the concentration of Fu-AuNPs was increased

424

from 25 to 100 µg mL-1. This revealed that the Fu-AuNPs are more effective in inhibiting the

425

viability of He La cells. When compared to bare chloroauric acid (HAuCl4) and fucoidan crude

426

extract, He La cells treated with Fu-AuNPs showed nuclear morphological changes such as cell

427

clumping and loss of membrane stability at 100 µg mL-1after 48 h. The cytotoxicity studies

428

revealed that Fu-AuNPs are promising in the growth of He La (cervical cancer) cells compared

429

to DMEM, saline, fucoidan crude extract and

430

accordance with the observations of Tengdelius et al. [38] who reported that the fucoidan-

431

mimetic glycopolymer showed cytotoxicity against human cancer colon cell line (HCT116).

432

Manju et al. [15] reported that NsEO-AuNPs effectively controlled the cell viability of A549

433

lung cancer cells at 50 µg mL-1. Hitherto, it has been reported that biogenic gold nanoparticles

434

synthesized using sargassum swartzii sea weed exhibits cytotoxic activity against He La cell

435

lines in a dose dependant manner [39]. The in vitro anticancer activity of AuNPs using Musa

436

paradisicica fruit peel extract against A-549 lung cancer cells has been previously reported by

437

Vijayakumar et al. [24].

438

The present study is aimed to search for a natural antimicrobial substance to replace antibiotics

439

for the treatment of A. hydrophila infections in fish. A. hydrophila is a fish specific pathogen that

440

causes fatal infection in fishes. The in vivo antibacterial activity of Fu-AuNPs was evaluated

441

against A. hydrophila using tilapia, O. mossambicus as model organism. Belemtougri et al. [40]

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bare chloroauric acid (HAuCl4). This was in

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reported that P. guajava leaf ethanol extracts were found to have a bacteriostatic effect on fish

443

pathogenic bacteria including A. hydrophila, A. salmonicida, Flavobacterium columnare,

444

Lactococcus garvieae, Streptococcus agalactiae and Vibrio salmonicida. Pachanawan et al. [41]

445

reported that P. guajava was added to a commercial fish diet either as a leaf powder or as a dried

446

ethanol leaf extract. Both supplements markedly reduced the mortality of tilapia experimentally

447

infected with A. hydrophila and no toxic effects were observed. In the present study, following

448

infection with A. hydrophila, O. mossambicus showed clinical symptoms like ulceration of skin,

449

hemorrhage. erythema on the external body surface such as mouth and operculum at pectoral fin

450

followed by fin rot at caudal fin. In addition, internal haemorrhage in the abdominal wall and

451

viscera were evident. Previously, it was reported that the platinum nanoparticles (PtNPs) exhibit

452

dose-dependent inhibition of bacterial proliferation and rescued zebrafish completely from the

453

bacteria infection [42]. Our results are supported by the observations of Thanigaivel et al. [43]

454

who reported that O. mossambicus infected with A. salmonicida exhibited haemorrhage at the

455

basal and oral fins. It was reported that the pathogenecity of A. salmonicida in swamp water

456

tilapia fishes, O. mossambicus exhibited ‘Furunculosis’ and high mortality of 80% Khatun et al.

457

[44]. The dietary supplementation of guajava leaf extract powder for 30 days significantly

458

reduced the mortality and increased the disease reisistance of O. mossambicus following in vivo

459

challenge with A. hydrophila in vivo at 107 cells/mL Gopi et al. [45].

460

5.0 Conclusion

461

The present study reports for the first time the synthesis and biophysical characterization of

462

fucoidan coated gold nanoparticles (Fu-AuNPs). In this study, Fu-AuNPs showed effective

463

inhibition of A. hydrophila at 100 µg mL-1 Furthermore, the biofilm of A. hydrophila was

464

completely arrested at 100 µg mL-1 of Fu-AuNPs in vitro. The in vivo challenge study clearly

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demonstrated that O. mossambicus infected with A. hydrophila exhibited haemorrrhage on the

466

basal and oral fins followed by abdominal wall and viscera. However, following administration

467

with Fu-AuNPs, a significant increase in the survival and better recovery from bacterial infection

468

was observed in O. mossambicus. This study concludes that Fu-AuNPs is a potential

469

antibacterial substance to fight bacterial infections and to improve the health status of fish.

470 471 472

Acknowledgement

473

under INSPIRE programme (INSPIRE Fellow-IF140145). The corresponding author

474

Dr.B.Vaseeharan thanks the Department of Biotechnology (DBT), New Delhi, India, for

475

financial assistance under the Project grants code: BT/PR7903/AAQ/3/638/2013.

476

Declaration of conflict of interest

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The authors report no conflicts of interest. References

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The first author S. Vijayakumar (SRF) thanks the DST, New Delhi, India for financial support

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on tilapia (Oreochromis mossambicus). J. Biosci. 15 (2007) 165–168.

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Benelli Oreochromis mossambicus diet supplementation with Psidium guajava leaf extracts

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enhance growth, immune, antioxidant response and resistance to Aeromonas hydrophila Fish &

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Shellfish Immunol 58 (2016) 572-583.

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Fig.1. UV–Vis spectrum of gold nanoparticles synthesized using Fucoidan crude extract

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Fig.2. XRD spectra showing various Bragg’s reflection peaks of gold nanoparticles synthesized using Fucoidan crude extract.

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Fig.6. EDX showing the elemental composition of gold nanoparticles synthesized using Fucoidan crude extract.

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Fig.8. (A) In vitro antibacterial activity of gold nanoparticles synthesized using fucoidan crude extract against A. hydrophila.

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Fig.8. (B) In vitro antibacterial activity of Fu-AuNPs against Aeromonas hydrophila in comparison with fucoidan crude extract, bare HuAuCl4 and chloramphenicol. Each bar indicated mean±standard deviations of three replications. Bars not labeled by the same letter represent statistical significance at P≤0.05 using ANOVA followed by Tukey’s HSD test.

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Fig.9. Microscopic image showing the antibiofilm activity of gold nanoparticles synthesized using Fucoidan crude extract against A. hydrophila at 40X magnification. (A) Light microscopy (B) Confocal laser scanning microscopy (2D view).

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Fig.10. (a) 3D view of confocal laser scanning microscopic image showing the thickness of bioilm growth of A. hydrophila Arrow indicates thick biofilm layer (in control) and thin biofilm layer (in treatment). (b) Reduction in biofilm thickness of bacteria after treatment with gold nanoparticles synthesized using Fucoidan crude extract at 100 µg mL-1. Each bar indicated mean±standard deviations of three replications. Bars not labeled by the same letter represent statistical significance at P≤0.05 using ANOVA followed by Tukey’s HSD test.

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Fig.11. Hydrophobicity index of A. hydrophila treated with gold nanoparticles synthesized using Fucoidan crude extract at 100 µg mL-1. Each bar indicated mean±standard deviations of three replications. Bars not labeled by the same letter represent statistical significance at P≤0.05 using ANOVA followed by Tukey’s HSD test.

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Fig.3. FTIR spectra showing functional groups of gold nanoparticles synthesized using Fucoidan crude extract.

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Fig.4. Zeta potential showing the surface charge of gold nanoparticles synthesized using Fucoidan crude extract.

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Fig.5.HR- TEM image of gold nanoparticles synthesized using Fucoidan crude extract.

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Fig. 7. Minimum inhibitory concentration (MIC) of Fu-AuNPs in comparison with fucoidan crude extract, bare HAuCl4 and chloramphenicol. Each bar indicated mean±standard deviations of three replications. Bars not labeled by the same letter represent statistical significance at P≤0.05 using ANOVA followed by Tukey’s HSD test.

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Fig.13. Protective effect of Fu-AuNPs on the survival of O. mossambicus infected with A. hydrophila. Each bar indicated mean±standard deviations of three replications. Bars not labeled by the same letter represent statistical significance at P≤0.05 using ANOVA followed by Tukey’s HSD test.

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Fig.12. (A) Cell viability of human cervical cancer cells (He LA cells) treated with Fu-AuNPs at different concentrations. Each bar indicated mean±standard deviations of three replications. Bars not labeled by the same letter represent statistical significance at P≤0.05 using ANOVA followed by Tukey’s HSD test. (B) Microscopic images showing the morphological changes in human cervical cancer cells (He LA cells) exposed to Fu-AuNPs at different concentrations (a) Phase contrast microscopy (b) Confocal laser scanning microscopy.

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Gold nanoparticles were synthesized using marine polysaccharide fucoidan from Fucus vesiculosus (Fu-AuNPs). Fu-AuNPs were physico-chemically characterized by UV-Vis spectroscopy, XRD, FTIR, HR-TEM, EDX and zeta potential. Fu-AuNPs exhibited antibacterial and antibiofilm activity against A. hydrophila. Fu-AuNPs showed anticancer activity against human cervical cancer (He La) cells. Fu-AuNPs significantly increased the survival of O. mossambicus following infection with A. hydrophila.