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Antimicrobial effects of chitosan silver nano composites (CAgNCs) on fish pathogenic Aliivibrio (Vibrio) salmonicida
Aquaculture 450 (2016) 422–430
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Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Antimicrobial effects of chitosan silver nano composites (CAgNCs) on fish pathogenic Aliivibrio (Vibrio) salmonicida S.H.S. Dananjaya a, G.I. Godahewa b, R.G.P.T. Jayasooriya b, Jehee Lee b,c, Mahanama De Zoysa a,c,⁎ a b c
College of Veterinary Medicine (BK21 Plus Program) and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea
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Article history: Received 17 August 2015 Accepted 19 August 2015 Available online 28 August 2015 Keywords: Chitosan silver nanocomposites Antibacterial agents Aliivibrio (Vibrio) salmonicida Reactive oxygen species Membrane permeability
a b s t r a c t Chitosan is one of the promising bio-degradable natural products which can be applied in nano forms in disease prevention, and treatment measures in aquaculture. The aim of this study was to develop and investigate the antibacterial function of chitosan-silver nano composites (CAgNCs) against fish pathogenic Aliivibrio salmonicida. Zeta potential and average size of synthesized CAgNCs were +32.1 mV and 281 nm, respectively. The Ag content of the CAgNCs was 0.643 ± 0.012% (w/w). Antibacterial results revealed that CAgNCs could inhibit the A. salmonicida growth which indicates minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) at 50 μg/mL and 100 μg/mL, respectively. We confirmed the attachment of AgNPs on the surface of A. salmonicida which indicates the interaction of CAgNCs with the bacterium. Propidium iodide (PI) uptake results suggested that CAgNCs has affected to permeability of cell membrane of A. salmonicida. Also, CAgNCs induced the level of reactive oxygen species (ROS) in concentration and time dependent manner (up to 3 h) suggesting that it may generate oxidative stress leading to bacterial cell death. Also, the level of protein was decreased in A. salmonicida cells after CAgNCs (50 μg/mL) treatment. DNA fragmentation assay results confirmed that CAgNCs can cause extensive DNA degradation of A. salmonicida which may inhibit the expression and production of bacterial proteins. Toxicity and safety results prove that CAgNCs is not toxic to zebrafish (Danio rerio) at 12.5 mg/kg of body weight/day as a feed ingredient and rock bream (Oplegnathus fasciatus) testis cells up to 50 μg/μL. Overall results from this study suggest that CAgNCs is potential antibacterial agent to control fish pathogenic bacteria. Statement of relevance: The fisheries and aquaculture industry can be optimized by using biodegradable nano materials such as chitosan for disease detection, control as well as delivery system of drugs such as hormones, vaccines and nutrients. Aquaculture can be optimized by using biodegradable nano materials such as chitosan for disease detection, control as well as delivery system of drugs such as hormones, vaccines and nutrients. Application of biodegradable nano materials in aquaculture © 2015 Elsevier B.V. All rights reserved.
1. Introduction During the past two decades, applications of metal nanoparticles (NPs) have been directed towards developing therapeutic agents with different bioactivities such as antimicrobial, wound healing, and anticancer. Metal nanoparticles as copper (Cu), gold (Au), and silver (Ag) have been widely tested for their biological properties (Mallick et al., 2012). Recently, Ag is considered as one of the most prominent and effective bactericidal agents in ionic form (Ag+1) as well as in nanoparticle (NP) state (Kim et al., 2011). AgNPs conjugation with antibiotic ⁎ Corresponding author at: College of Veterinary Medicine (BK21 Plus Program) and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea. E-mail address: [email protected] (M. De Zoysa).
http://dx.doi.org/10.1016/j.aquaculture.2015.08.023 0044-8486/© 2015 Elsevier B.V. All rights reserved.
prevents the antimicrobial resistance and enhances antimicrobial properties of the antibiotics (Raja and Singh, 2013). However, application of AgNPs itself should be strictly controlled due to the possibility of their accumulation in the tissues over the time that could lead toxic effects to host cells (You et al., 2012). Regiel et al. (2013) suggested that composite form of AgNPs with polymer based matrix such as chitosan could be a better option to enhance antimicrobial properties at lower concentrations that reduces the toxic effects. Thus, it is important to develop biodegradable nanocomposites with minimum adverse effect on host and the environment. Chitosan is a cationic biocompatible polysaccharide. It has been used to develop wide array of nano composites which has higher antibacterial activities (Akmaz et al., 2013) and immune stimulatory properties (Meshkini et al., 2012). AgNPs can be included in to chitosan matrix as nano filler using silver nitrate (AgNO3) as a precursor. Chitosan has
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ability to bind metal ions via chelation with the amine groups and used as a mild reducing agent for reduction of Ag ions. It is frequently employed as an ion capping agent to control the growth of nanoparticles and avoid their aggregation (Gracia et al., 2013). Most importantly, positively charged chitosan matrix can capture the negatively charged bacteria surface and AgNPs create pores on bacterial cell wall which cause rapid disintegration and alteration of membrane permeability towards cell death (Lo et al., 2013). Therefore, application of CAgNCs would be a better choice for controlling fish pathogens and other applications such as wound healing and drug delivery agents in aquaculture (Panyam and Labhasetwar, 2003). Moreover, it is essential to understand the mode of action and toxicity levels of CAgNCs before the application in the field level. Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the marine bacteria, Aliivibrio salmonicida (formerly known as Vibrio salmonicida). A. salmonicida shows a high survival rate under high salinity levels of ocean environment. Numbers of A. salmonicida colonies were ranged from 12 to 43 bacteria/mL in marine water, where concentrations being highest during the winter period (Enger et al., 1990). Therefore, considering future applications in aquaculture, we have used A. salmonicida as a model bacterium to understand the antibacterial effects of CAgNCs. In this study, stable CAgNCs were synthesized by reduction method and characterized its physiochemical properties. Antibacterial activities were investigated using agar diffusion and turbidimetric assays and determined the values of MIC and MBC. Additionally, FE-SEM image analysis was conducted to confirm the effects of CAgNCs on cell structure. Change of cell viability, ROS production, protein expression, DNA degradation and DNA binding capacity were investigated to understand the functional role of CAgNCs on bactericidal effects. Cytotoxicity of CAgNCs was tested by comparing cell viability and level of ROS using rock bream (Oplegnathus fasciatus) epithelial cells. 2. Material and method 2.1. Synthesis and characterization of CAgNCs CAgNCs was synthesized by reduction method as described previously (Dananjaya et al., 2014). In order to characterize the CAgNCs, physiochemical properties were investigated by determining UV–visible spectra using a spectrophotometer (Mecasys, Korea), particle size and zeta potential by Zetasizer Nano-ZS90 (Malvern Instruments, UK). Functional groups were assessed by Fourier Transforms Infrared (FTIR) spectrometer (Bio Rad, USA). The surface morphology was studied using FE-SEM analysis (Hitachi S-4800, Japan) operating at an accelerating voltage of 5.0 kV. The percentage of Ag in CAgNCs was determined using an Inductively Coupled Plasma–Atomic Emission Spectrometer (ICP–AES) (Perkin-Elmer Optima, USA). 2.2. Antibacterial activities of CAgNCs In order to investigate the antibacterial activity of CAgNCs, Gram negative A. salmonicida strain (KCTC 2766) was used in this study. A single colony of A. salmonicida was inoculated into 4 mL of marine broth (Becton, Dickinson, USA), and incubated at 25 °C under shaking (160 rpm) for 16 h. As a preliminary test, antimicrobial activity of CAgNCs was evaluated by the agar disk diffusion method (Sonawane et al., 2012). Tested range (0, 12.5, 25, 50, 75 μg/disc) of CAgNCs was dissolved in 0.25% (v/v) acetic acid. To determine the MIC and MBC, final concentrations of 0, 12.5, 25.0, 50.0, 75.0, and 100.0 μg/mL CAgNCs were added to 12 ml of marine broth with tested bacterial concentrations of 103–104 CFU mL−1 (CFU = Colony-Forming Units). The media were incubated at 25 °C with shaking at 160 rpm for 24 h in a shaking incubator. The MIC was determined by the measuring O.D. at 600 nm. Spread plate technique was used to determine the MBC of CAgNCs against A. salmonicida. Briefly, 100 μL of CAgNCs (0, 12.5, 25.0, 50.0,
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75.0, and 100 μg/ml) treated bacterial cultures were inoculated on marine agar plates. These samples were incubated at 25 °C for 24–36 h, and then colonies were quantified. Microdilution technique was used to determine the time series bacterial growth inhibition. CAgNCs with MA as a blank, culture with MA as a negative control, culture with ampicillin (50 μg/μL) as a positive control and culture with different concentrations of CAgNCs (12.5, 25.0, 50.0, 75.0, 100.0 μg/mL) were taken as experimental groups. 2.3. Field emission scanning electron microscope (FE-SEM) analysis To observe the morphological changes of A. salmonicida upon CAgNCs treatment, FE-SEM analysis was conducted. A. salmonicida cells (10 7 CFU mL − 1 ) were treated with CAgNCs (12.5, 50 and 75 μg/mL) for 6 h, and then centrifuged at 3500 rpm for 30 min. The pellets were washed using phosphate buffered saline (PBS) and pre-fixed with 2.5% glutaraldehyde for 30 min. The pre-fixed cells were washed by PBS and serially dehydrated using 30, 50, 70, 80, 90 and 100% ethanol. The fixed cells were dried and coated with osmium using ion sputter (E-1030, Hitachi, Japan). Treated samples were observed by FE-SEM (S-4800, Hitachi, Japan). Additionally, FE-SEM coupled with energy dispersive X-ray spectroscopy (FE-SEM JSM 7000 F-EDS, USA) was used to confirm the quantitative chemical composition on the selected areas of bacterial membranes. 2.4. Analysis of CAgNCs effects on ROS production of A. salmonicida In order to determine the effect of CAgNCs on ROS production, A. salmonicida was treated in concentration and time dependent manner. Briefly, A. salmonicida overnight culture was inoculated with fresh marine broth in 1:100. It was further incubated at 25 °C until reached up to 0.5 OD at 600 nm. Then, culture (4 mL) was treated by different concentrations of CAgNCs (6.25 to 75 μg/mL). Treated samples were incubated in a shaking incubator for 3 h at 25 °C. Then, cells were stained with 5-(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) at 30 μg/mL for 30 min followed by centrifugation at 13,000 rpm for 2 min to collect the cells. PBS washed cells were resuspended in PBS. ROS level was determined using FACScaliber flow cytometer (Becton Dickinson, USA). To find out the optimum incubation time for maximum ROS generation A. salmonicida was treated at 25 μg/μL CAgNCs and analyzed at different time intervals (1 h, 2 h, 3 h and 4 h). 2.5. Determination of bacterial cell viability Cell viability was determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, 500 μL (0.3 OD at 600 nm) of A. salmonicida culture was used in every treatment. Negative control, positive control (as mentioned in Section 2.2) and different concentrations of CAgNPs (6.25, 12.5, 18.75, 25, 50, 75 μg/μL) were used as the treatments. After 24 h incubation period, the samples were treated with 70 μg/μL of MTT solution and incubated for further 30 min. Harvested cells were resuspended in DMSO (200 μg/μL well) and cell viability was detected at OD570 nm using a micro plate reader (Thermo, USA). 2.6. Analysis of the effect of CAgNCs on cell membrane integrity by PI uptake Cell membrane integrity of CAgNCs treated bacteria was assessed by monitoring PI uptake (Sigma Aldrich, USA). Briefly, cell suspension of the control and CAgNCs (50 μg/μL) treated sample were centrifuged (3500 rpm) for 2 min and pellets were re-suspended in PBS. The cell suspensions were incubated with PI (4 μg/mL) at 25 °C for 15 min in dark. Cells were washed twice by PBS to remove unbound PI. An aliquot of each of these suspensions was placed on the cover slip and observed under an inverted fluorescence microscope (Leica DM5000 B, Germany)
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using 100 oil immersion objective. PI stained cells were viewed using 546 nm excitation and 620 nm emission filters and images were recorded (Leica DC300 FX, Leica Microsystems, and Switzerland). 2.7. Effects of CAgNCs on protein expression of bacterial cells Overnight bacterial culture was inoculated in to 20 mL of marine broth at 1:100 dilutions. When the bacterial growth reached to the 0.5 OD (600 nm), cells were harvested by centrifugation in 3500 rpm at 4 °C for 10 min. Then, pelleted cells were washed and re-suspended in 0.5% NaCl solution. The final cell suspension was adjusted to have an absorbance of 0.5 OD at 600 nm. To determine the time dependent effect of CAgNCs on protein synthesis, the 50 μg/mL CAgNCs was treated in different time intervals (30, 60, 120 and 180 min). Aliquots of 1 mL culture was withdrawn after 30, 60, 120 and 180 min and centrifuged. Samples obtained at different time intervals were subjected to 12% SDS–PAGE according to Sahu et al. (2009). The 0.05% Coomassie blue R-250 was used to stain the gel, followed by a standard de-staining procedure to the gel. 2.8. Analysis of CAgNCs induced DNA damage and binding capacity Different concentrations of CAgNCs (12.5, 25, 50 and 75 μg/mL) were treated as explained in Section 2.7. Treated samples were incubated at 25 °C for 6 h. Genomic DNA of A. salmonicida was extracted from CAgNCs treated and non-treated bacterial cells using the genomic DNA isolation kit (GeNet Bio, Korea). The isolated DNA (10 μL) was analyzed by standard agarose gel electrophoresis. To assess the in vitro DNA binding capacity of CAgNCs, overnight bacterial culture was subjected to isolate genomic DNA using genomic DNA isolation kit (GeNet Bio, Korea). Briefly, effect of CAgNCs on genomic DNA was performed by incubating the different amount of CAgNCs (0.5, 1.0, 1.5 and 2.0 μg) with 6 μg of isolated genomic DNA at 25 °C for 15 min. Formation of genomic DNA − CAgNCs complex was determined by 0.1% agarose gel electrophoresis. 2.9. Safety assessment of CAgNCs for fish Toxicity and safety level of CAgNCs was evaluated using zebrafish (in vivo) and rock bream testis cells (in vitro). Firstly, toxicity of CAgNCs was investigated using adult zebrafish by two administrative routes namely water bath exposure, and oral (feed). Briefly, zebrafish (body weight (0.4–0.5 g) were purchased from a local pet shop and acclimatized for 1 week under laboratory conditions at 26–28 °C with a 14 h: 10 h light–dark cycle. In water bath exposure, fish (n = 6) were exposed to concentrations of CAgNCs (250, 500, 750, 1000 and 1250 μg/L) in static tanks for 96 h. This experiment was conducted to three times and average value was used to calculate the 96-h LC50. In oral administration, CAgNCs were grinded and mixed with commercial fish diet. Fish (n = 30) were fed at 12.5 mg/kg of body weight per day (4% feed intake) for 14 days. Control fish were fed using commercial diet without CAgNCs. Once treatment commenced in all the experiments, fish were observed at 24 h intervals for mortality. In vivo toxicity of CAgNCs was investigated by determining cell viability and ROS production using cultured cells isolated from rock bream testis as described in previous study (Wan et al., 2012). Briefly, rock bream testis tissues were collected under aseptic conditions and minced into small pieces. Then subsequent washing in HBSS (sigma) containing antibiotics and followed by, the tissues were digested in a solution of 0.2% collagenase II (sigma). Digested tissue was filtered, centrifuged and re-suspended in Leibovitz's L-15 medium supplemented with FBS, penicillin and streptomycin. Sub-cultured rock bream testis cells (1 × 105 cells/well) were treated with different concentrations of CAgNCs for 24 h and cell viability was determined by MTT assay. In order to determine the ROS generation, testis cells were seeded at 5 × 106 cells/well and treated with different concentrations of CAgNCs.
Untreated and 0.25% acetic acid treated cells (negative control) were used as respective controls. All treated and untreated samples were incubated at 25 °C for 3 h. Then, H2DCFDA (30 μg/mL) was added into treated cells and incubated for 30 min. Cells were harvested by centrifugation (13,000 rpm for 2 min) and re-suspended in 500 μL of PBS. Level of ROS in rock bream testis cells was measured using the FACScaliber flow cytometer (Becton Dickinson, USA). 2.10. Statistical analysis All the data related to disc diffusion assay and cell viability were illustrated as means ± S.D. for triplicate reactions. Statistical analysis was performed using unpaired, two-tailed t-test to calculate the P-value using GraphPad program (GraphPad Software, Inc.). The significant difference was defined at P b 0.05. 3. Results 3.1. Synthesis and characterization of the CAgNCs As a first step, CAgNCs were synthesized using reduction method and characterized for their physiochemical properties. Initially, formation of CAgNCs was confirmed by observing the color change of chitosan-Ag solution from colorless to light yellow and then to yellowish brown within 10 min after adding 0.3 M NaOH. Synthesis of CAgNCs by reducing the silver ion into silver atom (Ag+1 → Ag°) was confirmed by UV–visible spectroscopy. The spectrum of chitosan solution, AgNO3 (0.01 M) and synthesized CAgNCs are shown in supplementary Fig. 1. As expected, only the CAgNCs showed a maximum absorbance (λ max) peak at ~ 415 nm which is a characteristic feature for AgNPs. None of the chitosan or AgNO3 (0.01 M) alone showed any peak for the entire spectral range of 275–650 nm. FT-IR analysis was conducted to detect functional groups which are responsible for the reduction of Ag+1, capping of newly synthesizing AgNPs, and potential bioactivities of CAgNCs. FT-IR spectrum analysis results showed the broad peak at 3427 cm−1 which corresponds to amine (− NH2) and hydroxyl (−OH) groups stretching vibration (supplementary Fig. 2). The amide (− CONH2) group and aliphatic C–H stretching were exhibited as peaks at 1658 cm−1 and 2877 cm−1, respectively. The zeta potential and average size of CAgNCs were +32.1 mV and 281 nm, respectively (supplementary Fig. 3). FE-SEM analysis results displayed that majority of AgNPs embedded in CAgNCs were in spherical shape and well dispersed in chitosan matrix (supplementary Fig. 4). The average size of the AgNPs was ranged between 15–35 nm and the calculated Ag content of CAgNCs was 0.643 ± 0.012% (w/w). 3.2. Antibacterial activity of CAgNCs against A. salmonicida We selected Gram negative marine bacterium A. salmonicida as a model to investigate bacteriostatic (bacteria-inhibiting) and bactericidal (bacteria killing) effects of CAgNCs. Agar disc diffusion and turbidimetric antibacterial assays were conducted to determine the MIC, MBC and time-kill profile with different concentrations of CAgNCs. Agar disc diffusion assay results revealed that CAgNCs inhibited the growth of A. salmonicida in a concentration-dependent manner and showed the highest growth arrest at 75 μg CAgNCs/disc after 24 h post incubation at 25 °C (Fig. 1). Interestingly, all treated concentrations of CAgNCs have shown significant antibacterial activity at P b 0.05. There was no inhibitory zone in the disc with 0.25% (V/V) acetic acid as negative control (solvent of CAgNCs). The ampicillin was used as positive control and 50 μg/ disc showed maximum inhibition of A. salmonicida (diameter of 20 mm) which is marginally higher than CAgNCs at 75 μg/disc (diameter of 20 mm). The turbidimetric antibacterial assay result provides the visible bacteriostatic concentrations of CAgNPs. The MIC and MBC of CAgNPs were 50 μg/mL and 100 μg/mL, respectively. The time-kill profile of A. salmonicida was constructed by measuring the growth of
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changes such as smooth cell margin. We observed that CAgNCs has caused intense changes in shape, appearance of pores like structures and damaged membrane integrity when treated with 12.5, 50.0, and 75.0 μg/mL concentrations. Treatment of 50 and 75 μg/mL of CAgNCs has resulted significant amount of cell debris, indicating that these concentrations are strong enough to induce a complete bacterial cell death (data not shown). Considering the central role of AgNPs in the regulation of bacterial cell lysis via changing the membrane permeability, we next attempted to quantitatively analyze the cell membrane elemental composition by FE-SEM equipped with an energy dispersive X-ray spectrum (EDS). Results clearly visualized the incorporation of AgNPs into the membrane of the bacteria cell (Fig. 3A). Fig. 3B shows the EDS analysis profile of bacterial cell membrane (selected) and it confirmed that cell membrane consists of about 19% of Silver (Ag), 41% of Carbon, 34% of oxygen and total 6% of other elements such as, Nitrogen (N), Sodium (Na), Chlorine (Cl), and Potassium (K). Fig. 1. The inhibition zone of CAgNCs against A. salmonicida. Different concentration (0–75 μg/disc) of CAgNCs was added on paper discs and plates were incubated at 25 °C for 24 h. The discs with 0.25% (V/V) acetic acid and ampicillin (50 μg/disc) were used as negative and positive controls, respectively. The diameter of the clear zone was measured in mm with diameter of disc (8 mm) for each treatment. The error bars indicate the mean ± S.D. (n = 3). Significant differences (P b 0.05) were calculated with respect to negative control (acetic acid). The asterisk (*) represent the significantly higher inhibition than control.
bacteria at 600 nm optical density after treated with CAgNCs. With the presence of 12.5, 25, 50 and 75 μg/mL of CAgNCs, the characteristic growth phases (lag, exponential, and stabilization) of A. salmonicida were changed when compared with untreated bacterial sample (control group) (Fig. 2). However, in absence of CAgNCs, A. salmonicida was able to reach exponential phase rapidly. All treated concentrations of CAgNCs caused a negative effect on A. salmonicida growth. Growth of the A. salmonicida cells, treated with 12.5 and 25 μg/mL CAgNCs were slightly lower than that of cells in the control group. The rapid and extensive bactericidal action of CAgNCs was clearly evidenced at 50 and 75 μg/ mL levels. When the concentration of CAgNCs was 50 μg/mL, there was no visible growth increment of A. salmonicida and therefore it was confirmed its MIC as 50 μg/mL.
3.4. Analysis of CAgNCs induced ROS production in A. salmonicida To measure the ROS production in CAgNCs treated A. salmonicida, flow cytometry analysis was done using 2, 7 dichlorofluorecinediacetate (H2DCFDA) as an intracellular ROS indicator. Results revealed that CAgNCs treated A. salmonicida can generate higher level of ROS in dose (6.25–75 μg/mL) and time dependent manner (Fig. 4). Untreated cells have shown lowest level (45.41) of ROS while 0.25% acetic acid (negative control) had slightly higher level (52.22) than that of untreated cells. ROS production was significantly increased from 6.25– 50 μg/mL of CAgNCs, however level of ROS at 50 μg/mL was slightly lower than 25 μg/mL. The highest level of ROS was found in cells treated at 25 μg/mL after 3 h. At the highest level of CAgNCs (75 μg/mL) showed the lowest ROS production among the all the CAgNCs concentrations. To investigate the time dependent ROS production, A. salmonicida were treated at 25 μg/mL of CAgNCs for 4 h. Result showed that ROS level was gradually increased until 3 h indicating the maximum level. Then, ROS level was decreased at 4 h but the level was higher than the untreated cells at 4 h (Fig. 4B). 3.5. Effect of CAgNCs on viability of A. salmonicida and PI uptake
3.3. Morphological changes of A. salmonicida of CAgNCs FE-SEM analysis was employed to monitor the morphological changes of A. salmonicida after the CAgNCs treatment. Untreated A. salmonicida exhibited typical rod shape and minimum morphological
A. salmonicida exposed to CAgNCs showed clear effect on cell viability as evident by the decrease in the formation of formazan in MTT assay (Fig. 5). The cell viability was significantly decreased when increasing CAgNCs concentration. However, highest cell viability (100%) was
Fig. 2. Growth inhibition profile of A. salmonicida after CAgNCs treatment. Bacterial cell growth was assessed after CAgNCs (0, 12.5, 25, 50 and 75 μg/mL) treatment at every 3 h intervals by measuring the OD at 600 nm. The bars indicate the mean ± S.D. (n = 3).
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Fig. 3. FE-SEM image with an energy dispersive X-ray spectrum (EDS). A) The FESEM image was 12.5 μg/mL CAgNCs treated A. salmonicida and incubated at 30 °C for 24 h before the preparation for FE-SEM. B) Energy dispersive X-ray spectrum of selected area of the bacteria cell.
noted at 6.25 μg/mL of CAgNCs. At the MIC concentration (50 μg/mL), cell viability was 50% whereas at 75 μg/mL, it was decreased up to 5%. The CAgNCs treated (50 μg/mL) A. salmonicida and untreated bacterial samples were stained with PI after the 4 h incubation. Stronger bright fluorescence was observed in the treated bacteria compared to untreated cells (supplementary Fig. 5). 3.6. Effect of CAgNCs on protein expression of A. salmonicida To investigate, CAgNCs effect on bacterial protein synthesis, whole cell protein expression was compared in treated (MIC-50 μg/mL) and untreated A. salmonicida at specific time intervals (30, 60 and 120 min) by SDS-PAGE. CAgNCs treated cells showed decreased level of protein at 30, 60 and 120 min compared to respective control samples (Fig. 6). However, there was no clear difference of protein levels at 30, 60 and 120 min after treatment of CAgNCs. 3.7. Effect of CAgNCs on the bacterial DNA To gather more insight into the mechanism of bacterial cell death after exposure to CAgNCs, we investigated a potential causal link between cell killing and DNA damage. DNA degradation and release of DNA fragments was analyzed by agarose gel electrophoresis by employing two methods of genomic DNA extraction, and genomic
DNA binding capacity methods, respectively. Analysis of DNA damage revealed that CAgNCs have depicted the potential DNA degradation in a dose dependent manner (Fig. 7A). There was no DNA damage upon 12.5 μg/mL CAgNCs treatment where it has shown similar amount of DNA to the control sample. However, 50 μg/mL and 75 μg/mL CAgNCs treatments have been shown significant DNA damage. Results of DNA binding analysis revealed that CAgNCs has potential to bind with genomic DNA of A. salmonicida and binding ability was increased in a dose dependent manner. The 0.5 and 1.0 μg of CAgNCs have shown less DNA binding activity, where 1.5 and 2.0 μg CAgNCs have shown complete binding with genomic DNA (Fig. 7B). 3.8. Safety assessment of CAgNCs using zebrafish and rock bream testis cells Laboratory tests were conducted to determine the safety of CAgNCs using zebrafish (in vivo) and rock bream testis cells (in vitro). Zebrafish showed 100% survival rate with CAgNCs based diet at 12.5 mg/kg of body weight/day (at 4% feed intake) for 14 days. We determined the LC 50 (96 h) of CAgNCs as 750 μg/L against zebrafish through water exposure at 25 °C. Preliminary results indicated that CAgNCs can be applied as water exposure and feed ingredient under given concentrations in zebrafish. However, application dose for other fish should be investigated. In vitro toxicity evaluation, CAgNCs treated rock bream testis cell line displayed almost equal cell viability upon the control and different
Fig. 4. Effect of CAgNCs on ROS production in A. salmonicida cells. Intracellular ROS generation was determined by the flow cytometry using H2DCFDA. A: concentration dependent ROS production; B: time dependent ROS production.
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Fig. 5. A. salmonicida cell viability detected by MTT assay. Bars represent the corresponding standard deviations (n = 3). Cell viability was assessed after treatment with different concentration of CAgNCs (6.25–75 μg/mL). The error bars indicate the mean ± S.D. (n = 3). Significant differences in A. salmonicida cell viability were calculated with respect to untreated control (P b 0.05). The * mark represent the significantly higher inhibition.
CAgNCs concentrations (Fig. 8). Furthermore, acetic acid also displayed similar cell viability but it is slightly lower than that of untreated control. To investigate the oxidative stress via intracellular ROS generation by CAgNCs, we performed the flow cytometric analysis using rock bream testis cells with CAgNCs. Results revealed that there was no distinguished intracellular ROS generation with the tested concentrations of CAgNCs; though they are statistically significant differ at P b 0.05. Therefore, CAgNCs may not create the oxidative stress for the fish cells (Fig. 9). 4. Discussion Present study was carried out to synthesize bio active CAgNCs and to investigate the antibacterial properties. As Twu et al. (2008) suggested degraded products of low-molecular weight chitosan (e.g., glucosamide) may provide electrons to act as a reducing agent. We used chitosan as reducing agent and stabilizer without any chemical reducing agent therefore, this method could be considered as ‘green synthesis’ approach to produce CAgNCs. The particle size and zeta potential of nano materials play an important role in antimicrobial performances (Abbaszadegan et al., 2014). They can alter particle stability and nano particle interaction
Fig. 6. Protein expression analysis of CAgNCs treated (MIC-50 μg/mL) A. salmonicida cells by SDS–PAGE. Cells were harvested 30, 60 and 120 min after treatment and analyzed for total protein. M: protein marker; Con: un-treated cells.
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with cell membrane. Comparatively small particle size and higher positive surface charge/zeta potential (+34 mv) in CAgNCs may have potential in antibacterial activities against Gram negative bacteria and other biological membranes. UV–vis absorption spectra results showed that characteristic SPR brand center of CAgNCs was between 410–420 nm (peak at ~415 nm). This range is within the SPR brand center of 417 nm of silver chitosan composite reported by Chen et al. (2014). It confirmed that CAgNCs contains AgNPs in chitosan matrix. FT-IR analysis provides the available functional groups and their comparison between chitosan and CAgNCs. The strong broad band at 3300–3500 cm−1 is characteristic of the N–H stretching in chitosan (Du et al., 2009). The peaks of amine group were shifted from 1658 cm−1 and 1597 cm−1 (pure chitosan) to 1641 cm−1 and 1560 cm−1 in CAgNCs corresponding to the bend vibration of amine chitosan (Bin et al., 2011). This suggests the attachment of Ag into N atoms (amino groups), which reduces the vibration intensity of the N–H bond due to greater molecular weight after binding of Ag atoms (Wei et al., 2009). The FT-IR spectrum of CAgNCs indicates the possibility of overlapping between the N–H and O–H stretching vibration. Overall results confirm synthesized CAgNCs consists of similar functional groups of normal chitosan in a different molecular size and bond arrangements which may attribute to higher therapeutic potential. Twu et al. (2008) described that the size of the AgNPs has been decreased, with increasing chitosan and sodium hydroxide concentrations. Therefore, varying concentration may give difference sizes of AgNPs. However, we applied 1% chitosan and 0.3 M sodium hydroxide concentration in the process and further studied are required to develop smaller size AgNPs. Characterization of CAgNCs revealed that our product could be used as potential antimicrobial agent and other biomedical applications. Investigation has done to confirm the antibacterial effects and understand the possible mode of action of CAgNCs, on cellular functions of A. salmonicida. Many other studies show antimicrobial properties of chitosan itself (Qi et al., 2004; Tao et al., 2011; You et al., 2012) and AgNPs (Du et al., 2009), however, relatively few attempts have been made to develop chitosan based Ag nano composites to obtain higher antibacterial properties and improve therapeutic power and of Ag with less side effects. It is interesting to develop chitosan based Ag nano composite in the present study and according to recent report by Chen et al. (2014), chitosan Ag nano composite has enhanced the antimicrobial activities against Escherichia coli, Salmonella choleraesuis, Staphylococcus aureus, and Bacillus subtilis compared to that of low molecular weight chitosan, however, MIC and MBC values are higher than 0.1 mg/mL for all the tested bacteria. Sanpui et al. (2008) showed MIC and MBC values of CAgNCs against E. coli were 100 and 120 μg/mL, respectively. Interestingly, MIC and MBC of the CAgNCs against A. salmonicida are lower (50 and 100 μg/mL) than previously reported chitosan based Ag nano composites, hence it could be suggested that CAgNCs may have stronger bactericidal effects against pathogenic bacteria. Additionally, A. salmonicida growth inhibition by CAgNCs (75 μg/mL) and ampicillin (50 μg/mL) has almost similar bactericidal activity. To demonstrate the mode of CAgNCs on the bactericidal effects, the changes in cell membrane (morphology and permeability), ROS production, state of bacterial chromosome (DNA fragmentation) and level of protein expression were compared in CAgNCs treated and control A. salmonicida. FE-SEM results confirmed the typical morphological changes such as irregular shape and ruptured cells with increasing concentration of CAgNCs. Additionally, we confirmed the attachment of AgNPs on the surface of cell wall which indicates the interaction of CAgNCs with the bacterium. The PI uptake is associated with cell membrane damage which indicates the alteration of cell membrane potential and it penetrates into cells and binds to DNA when the cells are compromised with high membrane porosity (Banerjee et al., 2010). Overall results from zeta potential, FE-SEM analysis combined with energy dispersive X-ray spectrum, PI uptake can suggest that positively charged CAgNCs has interacted with negatively charged A. salmonicida which caused the irreversible cell membrane damage leading to leakage of intracellular components.
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Fig. 7. DNA damage and binding activity of CAgNCs. (A) DNA damage analysis by agarose gel electrophoresis of genomic DNA samples extracted from A. salmonicida after CAgNCs treatment. 1: 0; 2:12.5 μg/mL, 3:25 μg/mL; 4: 50 μg/mL; 5: 75 μg/mL of CAgNCs. (B) DNA binding analysis by agarose gel electrophoresis of isolated genomic DNA (6 μg) of A. salmonicida treated with different amount CAgNCs. 1:1 Kb marker; 2: control genomic DNA; 3: 0.5 μg; 4: 1 μg; 5: 1.5 μg of CAgNCs.
Several reports have shown that bactericidal antibiotics promote the generation of ROS in E. coli which caused drug induced killing (Dwyer et al., 2012; Kohanski et al., 2008). Similarly AgNPs have been found to induce intracellular ROS levels (Su et al., 2009; You et al., 2012). On the other hand, AgNPs in the composite also can generate Ag+ and release into bacterial cell and interacts with thiol groups in proteins, resulting in inactivation of respiratory enzymes and leading to the production of ROS (Matsumura et al., 2003). Inoue et al. (2002) has described that ROS (eg. superoxide anion, hydrogen peroxide, hydroxyl radical, and singlet oxygen) contributed to the antibacterial activity against E. coli. Moreover, excessive levels of ROS can lead to damage DNA and inhibition or lowering the protein expression via oxidative stress (Marambio and Hoek, 2010). Mallick et al. (2012). Banerjee et al. (2010) documented the induction of ROS production in E. coli cell by iodinated chitosan-silver nano composite and iodine-stabilized chitosan Cu nano composite, respectively. Present study show that CAgNCs treatment generates higher ROS levels in A. salmonicida in concentration and time dependent manner. Also, results indicate the ROS induction at very low concentration (6.25 μg/μL) of CAgNCs while it maintains at higher and constant level at 50 and 75 μg/μL, respectively. The high ROS level correlates with the respective concentration of CAgNCs (50 μg/μL) at MIC level. However, ROS level was decreased (compared to 50 μg/μL) at concentration of 75 μg/μL which could be
Fig. 8. Effect of CAgNCs on ROS production in rock bream cells. Concentration dependent intracellular ROS generation was determined by the flow cytometry using H2DCFDA.
due to higher bacterial cell death. This is further supported by no any bacterial growth at 100 μg/μL which is the MBC of CAgNCs. MTT cell viability results indicated that cell viability was significantly decreased with increasing CAgNCs concentration showing 50% viable cells at 50 μg/μL which validate the MIC level of CAgNCs against A. salmonicida. It was shown that Ag+ ions prevent DNA replication and affect the structure and permeability of the cell membrane (Feng et al., 2000). Moreover, chitosan decreased the level of protein expression of S. aureus which may be due to inhibited protein synthesis or control gene expression (Tao et al., 2011; Xing et al., 2009). It reveals decreased protein expression in CAgNPs (50 μg/mL) treated A. salmonicida cells suggesting that CAgNPs could inhibit the protein synthesis. There was no clear difference of total protein under time series analysis up to 120 min and this could be due to immediate effect of CAgNCs on inactivation of protein expression (before 30 min). Furthermore, lower amount of total protein in CAgNCs treated bacteria cells could be due to loss of soluble protein as a result of membrane permeability and disrupting cell membranes as confirmed by PI and SEM analysis. Based on this, we propose that CAgNCs may regulate the inhibition of bacterial gene or protein expression in A. salmonicida and which mechanism needs to be elucidated in future. To understand the interaction between bacterial DNA and CAgNCs, the DNA binding and DNA damage assays were performed. Results confirmed that CAgNCs can strongly attach to genomic DNA of A. salmonicida in cells as well as in isolated form. It may be due to adhesion of bacterial DNA with composite through electrostatic interactions and subsequent creation of CAgNCs-DNA complex. This results is partially supported by previously study of iodine-stabilized Cu nanoparticle chitosan composite and iodinated chitosan-silver nanoparticle composite which binds the plasmid DNA of E. coli bacteria (Banerjee et al., 2010). In addition, chitosan composite binding with DNA have been reported the inhibition of mRNA synthesis of the bacteria (Dutta et al., 2009). To understand the role of CAgNCs on DNA fragmentation, we examined effect of CAgNCs on DNA degradation and it showed CAgNCs at MIC level can cause extensive cleavage of double-strand DNA that may lead to complete breakdown of genomic DNA. Thus, the complete DNA degradation appeared only at bactericidal concentration. The DNA degradation by different CAgNCs concentrations is correlated with cell death of A. salmonicida. Application of CAgNCs in bacterial control or any other therapeutic application in aquaculture, toxicity assessment is essential. Therefore, toxicity levels of CAgNCs were investigated using zebrafish (Water exposure
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Fig. 9. Rock bream cell viability detected by MTT assay. Bars represent the corresponding standard deviations (n = 3). Cell viability was assessed after treatment with different concentration of CAgNCs (6.25 μg/mL, 12.5 μg/mL, 18.75 μg/mL, 25 μg/mL and 50 μg/mL 75 μg/mL). The error bars indicate the mean ± S.D. (n = 3). Significant differences in rock bream testis cell viability were calculated with respect to untreated control (P b 0.05). The * mark represent the significantly higher inhibition.
and oral administration) and rock bream testis cells (cell viability and measuring the ROS production). Results prove that CAgNCs is not toxic to zebrafish at 12.5 mg/kg of body weight/day as a feed ingredient and rock bream testis cells up to 50 μg/μL. In conclusion, we synthesized the AgNPs embedded chitosan based CAgNCs which can effectively inhibit the fish pathogenic A. salmonicida. The cumulative effects of CAgNCs on physiological changes in cell membrane can be led to increase the cell membrane permeability and loss of cellular content in bacteria cells. Moreover, gradual release of AgNPs from chitosan matrix may promote the ROS production which can accelerate the DNA fragmentation, inhibition of protein expression and also halt the DNA repair and cellular homeostasis thereby causing for bacterial cell death. Therefore, application of significantly low toxic concentration of AgNPs in chitosan matrix in the form of composite (CAgNCs) is a much superior antibacterial agent in comparison to the use of antibiotics. Moreover, presence of chitosan may have direct effect on immune activation in fish, hence it could be considered as potential immune stimulant and vaccine adjuvant in aquaculture. Acknowledgments This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2014R1A2A1A11054585). Dananjaya S.H.S. is supported by BK21 Plus Program, Ministry of Education, Science & Technology (MEST), Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aquaculture.2015.08.023. References Abbaszadegan, A., Ghahramani, Y., Gholami, A., Hemmateenejad, B., Dorostkar, S., Nabavizadeh, M., Sharghi, S., 2014. The effect of charge at the surface of silver nanoparticles on antimicrobial activity against gram-positive and gram-negative bacteria: a preliminary study. J. Nanomater., 720654 Akmaz, S., Adjgüzel, E.D., Yasar, M., Erguven, O., 2013. The effect of Ag content of the chitosan-silver nanoparticle composite material on the structure and antibacterial activity. Adv. Mater. Sci. Eng., 690910 Banerjee, M., Mallick, S., Paul, A., Chattopadhyay, A., Ghosh, S.S., 2010. Heightened reactive oxygen species generation in the antimicrobial activity of a three component iodinated chitosan-silver nanoparticle composite. J. Surf. Colloids 26 (8), 5901–5908. Bin, A.M., Lim, J.J., Shameli, K., Ibrahim, N.A., Tay, M.Y., 2011. Synthesis of silver nanoparticles in chitosan, gelatin and chitosan/gelatin bionanocomposites by a chemical reducing agent and their characterization. Molecules 16 (9), 7237–7248. Chen, Q., Jiang, H., Ye, H., Li, J., Huang, J., 2014. Preparation, antibacterial, and antioxidant activities of silver/chitosan composites. J. Carbohydr. Chem. 1–15. Dananjaya, S.H.S., Godahewa, G.I., Jayasooriya, R.G.P.T., Chulhong, O.H., Lee, J., De Zoysa, M., 2014. Chitosan silver nano composites (CAgNCs) as potential antibacterial agent to control Vibrio tapetis. J. Vet. Sci. Technol. 5 (5), 209. Du, W.L., Niu, S.S., Xu, Y.L., Xu, Z.R., Fan, C.L., 2009. Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions. Carbohydr. Polym. 75 (3), 385–389.
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Aquaculture journal homepage: www.elsevier.com/locate/aqua-online
Antimicrobial effects of chitosan silver nano composites (CAgNCs) on fish pathogenic Aliivibrio (Vibrio) salmonicida S.H.S. Dananjaya a, G.I. Godahewa b, R.G.P.T. Jayasooriya b, Jehee Lee b,c, Mahanama De Zoysa a,c,⁎ a b c
College of Veterinary Medicine (BK21 Plus Program) and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea Department of Marine Life Sciences, School of Marine Biomedical Sciences, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea Fish Vaccine Research Center, Jeju National University, Jeju Self-Governing Province 690-756, Republic of Korea
a r t i c l e
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Article history: Received 17 August 2015 Accepted 19 August 2015 Available online 28 August 2015 Keywords: Chitosan silver nanocomposites Antibacterial agents Aliivibrio (Vibrio) salmonicida Reactive oxygen species Membrane permeability
a b s t r a c t Chitosan is one of the promising bio-degradable natural products which can be applied in nano forms in disease prevention, and treatment measures in aquaculture. The aim of this study was to develop and investigate the antibacterial function of chitosan-silver nano composites (CAgNCs) against fish pathogenic Aliivibrio salmonicida. Zeta potential and average size of synthesized CAgNCs were +32.1 mV and 281 nm, respectively. The Ag content of the CAgNCs was 0.643 ± 0.012% (w/w). Antibacterial results revealed that CAgNCs could inhibit the A. salmonicida growth which indicates minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) at 50 μg/mL and 100 μg/mL, respectively. We confirmed the attachment of AgNPs on the surface of A. salmonicida which indicates the interaction of CAgNCs with the bacterium. Propidium iodide (PI) uptake results suggested that CAgNCs has affected to permeability of cell membrane of A. salmonicida. Also, CAgNCs induced the level of reactive oxygen species (ROS) in concentration and time dependent manner (up to 3 h) suggesting that it may generate oxidative stress leading to bacterial cell death. Also, the level of protein was decreased in A. salmonicida cells after CAgNCs (50 μg/mL) treatment. DNA fragmentation assay results confirmed that CAgNCs can cause extensive DNA degradation of A. salmonicida which may inhibit the expression and production of bacterial proteins. Toxicity and safety results prove that CAgNCs is not toxic to zebrafish (Danio rerio) at 12.5 mg/kg of body weight/day as a feed ingredient and rock bream (Oplegnathus fasciatus) testis cells up to 50 μg/μL. Overall results from this study suggest that CAgNCs is potential antibacterial agent to control fish pathogenic bacteria. Statement of relevance: The fisheries and aquaculture industry can be optimized by using biodegradable nano materials such as chitosan for disease detection, control as well as delivery system of drugs such as hormones, vaccines and nutrients. Aquaculture can be optimized by using biodegradable nano materials such as chitosan for disease detection, control as well as delivery system of drugs such as hormones, vaccines and nutrients. Application of biodegradable nano materials in aquaculture © 2015 Elsevier B.V. All rights reserved.
1. Introduction During the past two decades, applications of metal nanoparticles (NPs) have been directed towards developing therapeutic agents with different bioactivities such as antimicrobial, wound healing, and anticancer. Metal nanoparticles as copper (Cu), gold (Au), and silver (Ag) have been widely tested for their biological properties (Mallick et al., 2012). Recently, Ag is considered as one of the most prominent and effective bactericidal agents in ionic form (Ag+1) as well as in nanoparticle (NP) state (Kim et al., 2011). AgNPs conjugation with antibiotic ⁎ Corresponding author at: College of Veterinary Medicine (BK21 Plus Program) and Research Institute of Veterinary Medicine, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea. E-mail address: [email protected] (M. De Zoysa).
http://dx.doi.org/10.1016/j.aquaculture.2015.08.023 0044-8486/© 2015 Elsevier B.V. All rights reserved.
prevents the antimicrobial resistance and enhances antimicrobial properties of the antibiotics (Raja and Singh, 2013). However, application of AgNPs itself should be strictly controlled due to the possibility of their accumulation in the tissues over the time that could lead toxic effects to host cells (You et al., 2012). Regiel et al. (2013) suggested that composite form of AgNPs with polymer based matrix such as chitosan could be a better option to enhance antimicrobial properties at lower concentrations that reduces the toxic effects. Thus, it is important to develop biodegradable nanocomposites with minimum adverse effect on host and the environment. Chitosan is a cationic biocompatible polysaccharide. It has been used to develop wide array of nano composites which has higher antibacterial activities (Akmaz et al., 2013) and immune stimulatory properties (Meshkini et al., 2012). AgNPs can be included in to chitosan matrix as nano filler using silver nitrate (AgNO3) as a precursor. Chitosan has
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ability to bind metal ions via chelation with the amine groups and used as a mild reducing agent for reduction of Ag ions. It is frequently employed as an ion capping agent to control the growth of nanoparticles and avoid their aggregation (Gracia et al., 2013). Most importantly, positively charged chitosan matrix can capture the negatively charged bacteria surface and AgNPs create pores on bacterial cell wall which cause rapid disintegration and alteration of membrane permeability towards cell death (Lo et al., 2013). Therefore, application of CAgNCs would be a better choice for controlling fish pathogens and other applications such as wound healing and drug delivery agents in aquaculture (Panyam and Labhasetwar, 2003). Moreover, it is essential to understand the mode of action and toxicity levels of CAgNCs before the application in the field level. Cold-water vibriosis (CV) is a bacterial septicemia of farmed salmonid fish and cod caused by the marine bacteria, Aliivibrio salmonicida (formerly known as Vibrio salmonicida). A. salmonicida shows a high survival rate under high salinity levels of ocean environment. Numbers of A. salmonicida colonies were ranged from 12 to 43 bacteria/mL in marine water, where concentrations being highest during the winter period (Enger et al., 1990). Therefore, considering future applications in aquaculture, we have used A. salmonicida as a model bacterium to understand the antibacterial effects of CAgNCs. In this study, stable CAgNCs were synthesized by reduction method and characterized its physiochemical properties. Antibacterial activities were investigated using agar diffusion and turbidimetric assays and determined the values of MIC and MBC. Additionally, FE-SEM image analysis was conducted to confirm the effects of CAgNCs on cell structure. Change of cell viability, ROS production, protein expression, DNA degradation and DNA binding capacity were investigated to understand the functional role of CAgNCs on bactericidal effects. Cytotoxicity of CAgNCs was tested by comparing cell viability and level of ROS using rock bream (Oplegnathus fasciatus) epithelial cells. 2. Material and method 2.1. Synthesis and characterization of CAgNCs CAgNCs was synthesized by reduction method as described previously (Dananjaya et al., 2014). In order to characterize the CAgNCs, physiochemical properties were investigated by determining UV–visible spectra using a spectrophotometer (Mecasys, Korea), particle size and zeta potential by Zetasizer Nano-ZS90 (Malvern Instruments, UK). Functional groups were assessed by Fourier Transforms Infrared (FTIR) spectrometer (Bio Rad, USA). The surface morphology was studied using FE-SEM analysis (Hitachi S-4800, Japan) operating at an accelerating voltage of 5.0 kV. The percentage of Ag in CAgNCs was determined using an Inductively Coupled Plasma–Atomic Emission Spectrometer (ICP–AES) (Perkin-Elmer Optima, USA). 2.2. Antibacterial activities of CAgNCs In order to investigate the antibacterial activity of CAgNCs, Gram negative A. salmonicida strain (KCTC 2766) was used in this study. A single colony of A. salmonicida was inoculated into 4 mL of marine broth (Becton, Dickinson, USA), and incubated at 25 °C under shaking (160 rpm) for 16 h. As a preliminary test, antimicrobial activity of CAgNCs was evaluated by the agar disk diffusion method (Sonawane et al., 2012). Tested range (0, 12.5, 25, 50, 75 μg/disc) of CAgNCs was dissolved in 0.25% (v/v) acetic acid. To determine the MIC and MBC, final concentrations of 0, 12.5, 25.0, 50.0, 75.0, and 100.0 μg/mL CAgNCs were added to 12 ml of marine broth with tested bacterial concentrations of 103–104 CFU mL−1 (CFU = Colony-Forming Units). The media were incubated at 25 °C with shaking at 160 rpm for 24 h in a shaking incubator. The MIC was determined by the measuring O.D. at 600 nm. Spread plate technique was used to determine the MBC of CAgNCs against A. salmonicida. Briefly, 100 μL of CAgNCs (0, 12.5, 25.0, 50.0,
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75.0, and 100 μg/ml) treated bacterial cultures were inoculated on marine agar plates. These samples were incubated at 25 °C for 24–36 h, and then colonies were quantified. Microdilution technique was used to determine the time series bacterial growth inhibition. CAgNCs with MA as a blank, culture with MA as a negative control, culture with ampicillin (50 μg/μL) as a positive control and culture with different concentrations of CAgNCs (12.5, 25.0, 50.0, 75.0, 100.0 μg/mL) were taken as experimental groups. 2.3. Field emission scanning electron microscope (FE-SEM) analysis To observe the morphological changes of A. salmonicida upon CAgNCs treatment, FE-SEM analysis was conducted. A. salmonicida cells (10 7 CFU mL − 1 ) were treated with CAgNCs (12.5, 50 and 75 μg/mL) for 6 h, and then centrifuged at 3500 rpm for 30 min. The pellets were washed using phosphate buffered saline (PBS) and pre-fixed with 2.5% glutaraldehyde for 30 min. The pre-fixed cells were washed by PBS and serially dehydrated using 30, 50, 70, 80, 90 and 100% ethanol. The fixed cells were dried and coated with osmium using ion sputter (E-1030, Hitachi, Japan). Treated samples were observed by FE-SEM (S-4800, Hitachi, Japan). Additionally, FE-SEM coupled with energy dispersive X-ray spectroscopy (FE-SEM JSM 7000 F-EDS, USA) was used to confirm the quantitative chemical composition on the selected areas of bacterial membranes. 2.4. Analysis of CAgNCs effects on ROS production of A. salmonicida In order to determine the effect of CAgNCs on ROS production, A. salmonicida was treated in concentration and time dependent manner. Briefly, A. salmonicida overnight culture was inoculated with fresh marine broth in 1:100. It was further incubated at 25 °C until reached up to 0.5 OD at 600 nm. Then, culture (4 mL) was treated by different concentrations of CAgNCs (6.25 to 75 μg/mL). Treated samples were incubated in a shaking incubator for 3 h at 25 °C. Then, cells were stained with 5-(6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) at 30 μg/mL for 30 min followed by centrifugation at 13,000 rpm for 2 min to collect the cells. PBS washed cells were resuspended in PBS. ROS level was determined using FACScaliber flow cytometer (Becton Dickinson, USA). To find out the optimum incubation time for maximum ROS generation A. salmonicida was treated at 25 μg/μL CAgNCs and analyzed at different time intervals (1 h, 2 h, 3 h and 4 h). 2.5. Determination of bacterial cell viability Cell viability was determined by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, 500 μL (0.3 OD at 600 nm) of A. salmonicida culture was used in every treatment. Negative control, positive control (as mentioned in Section 2.2) and different concentrations of CAgNPs (6.25, 12.5, 18.75, 25, 50, 75 μg/μL) were used as the treatments. After 24 h incubation period, the samples were treated with 70 μg/μL of MTT solution and incubated for further 30 min. Harvested cells were resuspended in DMSO (200 μg/μL well) and cell viability was detected at OD570 nm using a micro plate reader (Thermo, USA). 2.6. Analysis of the effect of CAgNCs on cell membrane integrity by PI uptake Cell membrane integrity of CAgNCs treated bacteria was assessed by monitoring PI uptake (Sigma Aldrich, USA). Briefly, cell suspension of the control and CAgNCs (50 μg/μL) treated sample were centrifuged (3500 rpm) for 2 min and pellets were re-suspended in PBS. The cell suspensions were incubated with PI (4 μg/mL) at 25 °C for 15 min in dark. Cells were washed twice by PBS to remove unbound PI. An aliquot of each of these suspensions was placed on the cover slip and observed under an inverted fluorescence microscope (Leica DM5000 B, Germany)
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using 100 oil immersion objective. PI stained cells were viewed using 546 nm excitation and 620 nm emission filters and images were recorded (Leica DC300 FX, Leica Microsystems, and Switzerland). 2.7. Effects of CAgNCs on protein expression of bacterial cells Overnight bacterial culture was inoculated in to 20 mL of marine broth at 1:100 dilutions. When the bacterial growth reached to the 0.5 OD (600 nm), cells were harvested by centrifugation in 3500 rpm at 4 °C for 10 min. Then, pelleted cells were washed and re-suspended in 0.5% NaCl solution. The final cell suspension was adjusted to have an absorbance of 0.5 OD at 600 nm. To determine the time dependent effect of CAgNCs on protein synthesis, the 50 μg/mL CAgNCs was treated in different time intervals (30, 60, 120 and 180 min). Aliquots of 1 mL culture was withdrawn after 30, 60, 120 and 180 min and centrifuged. Samples obtained at different time intervals were subjected to 12% SDS–PAGE according to Sahu et al. (2009). The 0.05% Coomassie blue R-250 was used to stain the gel, followed by a standard de-staining procedure to the gel. 2.8. Analysis of CAgNCs induced DNA damage and binding capacity Different concentrations of CAgNCs (12.5, 25, 50 and 75 μg/mL) were treated as explained in Section 2.7. Treated samples were incubated at 25 °C for 6 h. Genomic DNA of A. salmonicida was extracted from CAgNCs treated and non-treated bacterial cells using the genomic DNA isolation kit (GeNet Bio, Korea). The isolated DNA (10 μL) was analyzed by standard agarose gel electrophoresis. To assess the in vitro DNA binding capacity of CAgNCs, overnight bacterial culture was subjected to isolate genomic DNA using genomic DNA isolation kit (GeNet Bio, Korea). Briefly, effect of CAgNCs on genomic DNA was performed by incubating the different amount of CAgNCs (0.5, 1.0, 1.5 and 2.0 μg) with 6 μg of isolated genomic DNA at 25 °C for 15 min. Formation of genomic DNA − CAgNCs complex was determined by 0.1% agarose gel electrophoresis. 2.9. Safety assessment of CAgNCs for fish Toxicity and safety level of CAgNCs was evaluated using zebrafish (in vivo) and rock bream testis cells (in vitro). Firstly, toxicity of CAgNCs was investigated using adult zebrafish by two administrative routes namely water bath exposure, and oral (feed). Briefly, zebrafish (body weight (0.4–0.5 g) were purchased from a local pet shop and acclimatized for 1 week under laboratory conditions at 26–28 °C with a 14 h: 10 h light–dark cycle. In water bath exposure, fish (n = 6) were exposed to concentrations of CAgNCs (250, 500, 750, 1000 and 1250 μg/L) in static tanks for 96 h. This experiment was conducted to three times and average value was used to calculate the 96-h LC50. In oral administration, CAgNCs were grinded and mixed with commercial fish diet. Fish (n = 30) were fed at 12.5 mg/kg of body weight per day (4% feed intake) for 14 days. Control fish were fed using commercial diet without CAgNCs. Once treatment commenced in all the experiments, fish were observed at 24 h intervals for mortality. In vivo toxicity of CAgNCs was investigated by determining cell viability and ROS production using cultured cells isolated from rock bream testis as described in previous study (Wan et al., 2012). Briefly, rock bream testis tissues were collected under aseptic conditions and minced into small pieces. Then subsequent washing in HBSS (sigma) containing antibiotics and followed by, the tissues were digested in a solution of 0.2% collagenase II (sigma). Digested tissue was filtered, centrifuged and re-suspended in Leibovitz's L-15 medium supplemented with FBS, penicillin and streptomycin. Sub-cultured rock bream testis cells (1 × 105 cells/well) were treated with different concentrations of CAgNCs for 24 h and cell viability was determined by MTT assay. In order to determine the ROS generation, testis cells were seeded at 5 × 106 cells/well and treated with different concentrations of CAgNCs.
Untreated and 0.25% acetic acid treated cells (negative control) were used as respective controls. All treated and untreated samples were incubated at 25 °C for 3 h. Then, H2DCFDA (30 μg/mL) was added into treated cells and incubated for 30 min. Cells were harvested by centrifugation (13,000 rpm for 2 min) and re-suspended in 500 μL of PBS. Level of ROS in rock bream testis cells was measured using the FACScaliber flow cytometer (Becton Dickinson, USA). 2.10. Statistical analysis All the data related to disc diffusion assay and cell viability were illustrated as means ± S.D. for triplicate reactions. Statistical analysis was performed using unpaired, two-tailed t-test to calculate the P-value using GraphPad program (GraphPad Software, Inc.). The significant difference was defined at P b 0.05. 3. Results 3.1. Synthesis and characterization of the CAgNCs As a first step, CAgNCs were synthesized using reduction method and characterized for their physiochemical properties. Initially, formation of CAgNCs was confirmed by observing the color change of chitosan-Ag solution from colorless to light yellow and then to yellowish brown within 10 min after adding 0.3 M NaOH. Synthesis of CAgNCs by reducing the silver ion into silver atom (Ag+1 → Ag°) was confirmed by UV–visible spectroscopy. The spectrum of chitosan solution, AgNO3 (0.01 M) and synthesized CAgNCs are shown in supplementary Fig. 1. As expected, only the CAgNCs showed a maximum absorbance (λ max) peak at ~ 415 nm which is a characteristic feature for AgNPs. None of the chitosan or AgNO3 (0.01 M) alone showed any peak for the entire spectral range of 275–650 nm. FT-IR analysis was conducted to detect functional groups which are responsible for the reduction of Ag+1, capping of newly synthesizing AgNPs, and potential bioactivities of CAgNCs. FT-IR spectrum analysis results showed the broad peak at 3427 cm−1 which corresponds to amine (− NH2) and hydroxyl (−OH) groups stretching vibration (supplementary Fig. 2). The amide (− CONH2) group and aliphatic C–H stretching were exhibited as peaks at 1658 cm−1 and 2877 cm−1, respectively. The zeta potential and average size of CAgNCs were +32.1 mV and 281 nm, respectively (supplementary Fig. 3). FE-SEM analysis results displayed that majority of AgNPs embedded in CAgNCs were in spherical shape and well dispersed in chitosan matrix (supplementary Fig. 4). The average size of the AgNPs was ranged between 15–35 nm and the calculated Ag content of CAgNCs was 0.643 ± 0.012% (w/w). 3.2. Antibacterial activity of CAgNCs against A. salmonicida We selected Gram negative marine bacterium A. salmonicida as a model to investigate bacteriostatic (bacteria-inhibiting) and bactericidal (bacteria killing) effects of CAgNCs. Agar disc diffusion and turbidimetric antibacterial assays were conducted to determine the MIC, MBC and time-kill profile with different concentrations of CAgNCs. Agar disc diffusion assay results revealed that CAgNCs inhibited the growth of A. salmonicida in a concentration-dependent manner and showed the highest growth arrest at 75 μg CAgNCs/disc after 24 h post incubation at 25 °C (Fig. 1). Interestingly, all treated concentrations of CAgNCs have shown significant antibacterial activity at P b 0.05. There was no inhibitory zone in the disc with 0.25% (V/V) acetic acid as negative control (solvent of CAgNCs). The ampicillin was used as positive control and 50 μg/ disc showed maximum inhibition of A. salmonicida (diameter of 20 mm) which is marginally higher than CAgNCs at 75 μg/disc (diameter of 20 mm). The turbidimetric antibacterial assay result provides the visible bacteriostatic concentrations of CAgNPs. The MIC and MBC of CAgNPs were 50 μg/mL and 100 μg/mL, respectively. The time-kill profile of A. salmonicida was constructed by measuring the growth of
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changes such as smooth cell margin. We observed that CAgNCs has caused intense changes in shape, appearance of pores like structures and damaged membrane integrity when treated with 12.5, 50.0, and 75.0 μg/mL concentrations. Treatment of 50 and 75 μg/mL of CAgNCs has resulted significant amount of cell debris, indicating that these concentrations are strong enough to induce a complete bacterial cell death (data not shown). Considering the central role of AgNPs in the regulation of bacterial cell lysis via changing the membrane permeability, we next attempted to quantitatively analyze the cell membrane elemental composition by FE-SEM equipped with an energy dispersive X-ray spectrum (EDS). Results clearly visualized the incorporation of AgNPs into the membrane of the bacteria cell (Fig. 3A). Fig. 3B shows the EDS analysis profile of bacterial cell membrane (selected) and it confirmed that cell membrane consists of about 19% of Silver (Ag), 41% of Carbon, 34% of oxygen and total 6% of other elements such as, Nitrogen (N), Sodium (Na), Chlorine (Cl), and Potassium (K). Fig. 1. The inhibition zone of CAgNCs against A. salmonicida. Different concentration (0–75 μg/disc) of CAgNCs was added on paper discs and plates were incubated at 25 °C for 24 h. The discs with 0.25% (V/V) acetic acid and ampicillin (50 μg/disc) were used as negative and positive controls, respectively. The diameter of the clear zone was measured in mm with diameter of disc (8 mm) for each treatment. The error bars indicate the mean ± S.D. (n = 3). Significant differences (P b 0.05) were calculated with respect to negative control (acetic acid). The asterisk (*) represent the significantly higher inhibition than control.
bacteria at 600 nm optical density after treated with CAgNCs. With the presence of 12.5, 25, 50 and 75 μg/mL of CAgNCs, the characteristic growth phases (lag, exponential, and stabilization) of A. salmonicida were changed when compared with untreated bacterial sample (control group) (Fig. 2). However, in absence of CAgNCs, A. salmonicida was able to reach exponential phase rapidly. All treated concentrations of CAgNCs caused a negative effect on A. salmonicida growth. Growth of the A. salmonicida cells, treated with 12.5 and 25 μg/mL CAgNCs were slightly lower than that of cells in the control group. The rapid and extensive bactericidal action of CAgNCs was clearly evidenced at 50 and 75 μg/ mL levels. When the concentration of CAgNCs was 50 μg/mL, there was no visible growth increment of A. salmonicida and therefore it was confirmed its MIC as 50 μg/mL.
3.4. Analysis of CAgNCs induced ROS production in A. salmonicida To measure the ROS production in CAgNCs treated A. salmonicida, flow cytometry analysis was done using 2, 7 dichlorofluorecinediacetate (H2DCFDA) as an intracellular ROS indicator. Results revealed that CAgNCs treated A. salmonicida can generate higher level of ROS in dose (6.25–75 μg/mL) and time dependent manner (Fig. 4). Untreated cells have shown lowest level (45.41) of ROS while 0.25% acetic acid (negative control) had slightly higher level (52.22) than that of untreated cells. ROS production was significantly increased from 6.25– 50 μg/mL of CAgNCs, however level of ROS at 50 μg/mL was slightly lower than 25 μg/mL. The highest level of ROS was found in cells treated at 25 μg/mL after 3 h. At the highest level of CAgNCs (75 μg/mL) showed the lowest ROS production among the all the CAgNCs concentrations. To investigate the time dependent ROS production, A. salmonicida were treated at 25 μg/mL of CAgNCs for 4 h. Result showed that ROS level was gradually increased until 3 h indicating the maximum level. Then, ROS level was decreased at 4 h but the level was higher than the untreated cells at 4 h (Fig. 4B). 3.5. Effect of CAgNCs on viability of A. salmonicida and PI uptake
3.3. Morphological changes of A. salmonicida of CAgNCs FE-SEM analysis was employed to monitor the morphological changes of A. salmonicida after the CAgNCs treatment. Untreated A. salmonicida exhibited typical rod shape and minimum morphological
A. salmonicida exposed to CAgNCs showed clear effect on cell viability as evident by the decrease in the formation of formazan in MTT assay (Fig. 5). The cell viability was significantly decreased when increasing CAgNCs concentration. However, highest cell viability (100%) was
Fig. 2. Growth inhibition profile of A. salmonicida after CAgNCs treatment. Bacterial cell growth was assessed after CAgNCs (0, 12.5, 25, 50 and 75 μg/mL) treatment at every 3 h intervals by measuring the OD at 600 nm. The bars indicate the mean ± S.D. (n = 3).
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Fig. 3. FE-SEM image with an energy dispersive X-ray spectrum (EDS). A) The FESEM image was 12.5 μg/mL CAgNCs treated A. salmonicida and incubated at 30 °C for 24 h before the preparation for FE-SEM. B) Energy dispersive X-ray spectrum of selected area of the bacteria cell.
noted at 6.25 μg/mL of CAgNCs. At the MIC concentration (50 μg/mL), cell viability was 50% whereas at 75 μg/mL, it was decreased up to 5%. The CAgNCs treated (50 μg/mL) A. salmonicida and untreated bacterial samples were stained with PI after the 4 h incubation. Stronger bright fluorescence was observed in the treated bacteria compared to untreated cells (supplementary Fig. 5). 3.6. Effect of CAgNCs on protein expression of A. salmonicida To investigate, CAgNCs effect on bacterial protein synthesis, whole cell protein expression was compared in treated (MIC-50 μg/mL) and untreated A. salmonicida at specific time intervals (30, 60 and 120 min) by SDS-PAGE. CAgNCs treated cells showed decreased level of protein at 30, 60 and 120 min compared to respective control samples (Fig. 6). However, there was no clear difference of protein levels at 30, 60 and 120 min after treatment of CAgNCs. 3.7. Effect of CAgNCs on the bacterial DNA To gather more insight into the mechanism of bacterial cell death after exposure to CAgNCs, we investigated a potential causal link between cell killing and DNA damage. DNA degradation and release of DNA fragments was analyzed by agarose gel electrophoresis by employing two methods of genomic DNA extraction, and genomic
DNA binding capacity methods, respectively. Analysis of DNA damage revealed that CAgNCs have depicted the potential DNA degradation in a dose dependent manner (Fig. 7A). There was no DNA damage upon 12.5 μg/mL CAgNCs treatment where it has shown similar amount of DNA to the control sample. However, 50 μg/mL and 75 μg/mL CAgNCs treatments have been shown significant DNA damage. Results of DNA binding analysis revealed that CAgNCs has potential to bind with genomic DNA of A. salmonicida and binding ability was increased in a dose dependent manner. The 0.5 and 1.0 μg of CAgNCs have shown less DNA binding activity, where 1.5 and 2.0 μg CAgNCs have shown complete binding with genomic DNA (Fig. 7B). 3.8. Safety assessment of CAgNCs using zebrafish and rock bream testis cells Laboratory tests were conducted to determine the safety of CAgNCs using zebrafish (in vivo) and rock bream testis cells (in vitro). Zebrafish showed 100% survival rate with CAgNCs based diet at 12.5 mg/kg of body weight/day (at 4% feed intake) for 14 days. We determined the LC 50 (96 h) of CAgNCs as 750 μg/L against zebrafish through water exposure at 25 °C. Preliminary results indicated that CAgNCs can be applied as water exposure and feed ingredient under given concentrations in zebrafish. However, application dose for other fish should be investigated. In vitro toxicity evaluation, CAgNCs treated rock bream testis cell line displayed almost equal cell viability upon the control and different
Fig. 4. Effect of CAgNCs on ROS production in A. salmonicida cells. Intracellular ROS generation was determined by the flow cytometry using H2DCFDA. A: concentration dependent ROS production; B: time dependent ROS production.
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Fig. 5. A. salmonicida cell viability detected by MTT assay. Bars represent the corresponding standard deviations (n = 3). Cell viability was assessed after treatment with different concentration of CAgNCs (6.25–75 μg/mL). The error bars indicate the mean ± S.D. (n = 3). Significant differences in A. salmonicida cell viability were calculated with respect to untreated control (P b 0.05). The * mark represent the significantly higher inhibition.
CAgNCs concentrations (Fig. 8). Furthermore, acetic acid also displayed similar cell viability but it is slightly lower than that of untreated control. To investigate the oxidative stress via intracellular ROS generation by CAgNCs, we performed the flow cytometric analysis using rock bream testis cells with CAgNCs. Results revealed that there was no distinguished intracellular ROS generation with the tested concentrations of CAgNCs; though they are statistically significant differ at P b 0.05. Therefore, CAgNCs may not create the oxidative stress for the fish cells (Fig. 9). 4. Discussion Present study was carried out to synthesize bio active CAgNCs and to investigate the antibacterial properties. As Twu et al. (2008) suggested degraded products of low-molecular weight chitosan (e.g., glucosamide) may provide electrons to act as a reducing agent. We used chitosan as reducing agent and stabilizer without any chemical reducing agent therefore, this method could be considered as ‘green synthesis’ approach to produce CAgNCs. The particle size and zeta potential of nano materials play an important role in antimicrobial performances (Abbaszadegan et al., 2014). They can alter particle stability and nano particle interaction
Fig. 6. Protein expression analysis of CAgNCs treated (MIC-50 μg/mL) A. salmonicida cells by SDS–PAGE. Cells were harvested 30, 60 and 120 min after treatment and analyzed for total protein. M: protein marker; Con: un-treated cells.
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with cell membrane. Comparatively small particle size and higher positive surface charge/zeta potential (+34 mv) in CAgNCs may have potential in antibacterial activities against Gram negative bacteria and other biological membranes. UV–vis absorption spectra results showed that characteristic SPR brand center of CAgNCs was between 410–420 nm (peak at ~415 nm). This range is within the SPR brand center of 417 nm of silver chitosan composite reported by Chen et al. (2014). It confirmed that CAgNCs contains AgNPs in chitosan matrix. FT-IR analysis provides the available functional groups and their comparison between chitosan and CAgNCs. The strong broad band at 3300–3500 cm−1 is characteristic of the N–H stretching in chitosan (Du et al., 2009). The peaks of amine group were shifted from 1658 cm−1 and 1597 cm−1 (pure chitosan) to 1641 cm−1 and 1560 cm−1 in CAgNCs corresponding to the bend vibration of amine chitosan (Bin et al., 2011). This suggests the attachment of Ag into N atoms (amino groups), which reduces the vibration intensity of the N–H bond due to greater molecular weight after binding of Ag atoms (Wei et al., 2009). The FT-IR spectrum of CAgNCs indicates the possibility of overlapping between the N–H and O–H stretching vibration. Overall results confirm synthesized CAgNCs consists of similar functional groups of normal chitosan in a different molecular size and bond arrangements which may attribute to higher therapeutic potential. Twu et al. (2008) described that the size of the AgNPs has been decreased, with increasing chitosan and sodium hydroxide concentrations. Therefore, varying concentration may give difference sizes of AgNPs. However, we applied 1% chitosan and 0.3 M sodium hydroxide concentration in the process and further studied are required to develop smaller size AgNPs. Characterization of CAgNCs revealed that our product could be used as potential antimicrobial agent and other biomedical applications. Investigation has done to confirm the antibacterial effects and understand the possible mode of action of CAgNCs, on cellular functions of A. salmonicida. Many other studies show antimicrobial properties of chitosan itself (Qi et al., 2004; Tao et al., 2011; You et al., 2012) and AgNPs (Du et al., 2009), however, relatively few attempts have been made to develop chitosan based Ag nano composites to obtain higher antibacterial properties and improve therapeutic power and of Ag with less side effects. It is interesting to develop chitosan based Ag nano composite in the present study and according to recent report by Chen et al. (2014), chitosan Ag nano composite has enhanced the antimicrobial activities against Escherichia coli, Salmonella choleraesuis, Staphylococcus aureus, and Bacillus subtilis compared to that of low molecular weight chitosan, however, MIC and MBC values are higher than 0.1 mg/mL for all the tested bacteria. Sanpui et al. (2008) showed MIC and MBC values of CAgNCs against E. coli were 100 and 120 μg/mL, respectively. Interestingly, MIC and MBC of the CAgNCs against A. salmonicida are lower (50 and 100 μg/mL) than previously reported chitosan based Ag nano composites, hence it could be suggested that CAgNCs may have stronger bactericidal effects against pathogenic bacteria. Additionally, A. salmonicida growth inhibition by CAgNCs (75 μg/mL) and ampicillin (50 μg/mL) has almost similar bactericidal activity. To demonstrate the mode of CAgNCs on the bactericidal effects, the changes in cell membrane (morphology and permeability), ROS production, state of bacterial chromosome (DNA fragmentation) and level of protein expression were compared in CAgNCs treated and control A. salmonicida. FE-SEM results confirmed the typical morphological changes such as irregular shape and ruptured cells with increasing concentration of CAgNCs. Additionally, we confirmed the attachment of AgNPs on the surface of cell wall which indicates the interaction of CAgNCs with the bacterium. The PI uptake is associated with cell membrane damage which indicates the alteration of cell membrane potential and it penetrates into cells and binds to DNA when the cells are compromised with high membrane porosity (Banerjee et al., 2010). Overall results from zeta potential, FE-SEM analysis combined with energy dispersive X-ray spectrum, PI uptake can suggest that positively charged CAgNCs has interacted with negatively charged A. salmonicida which caused the irreversible cell membrane damage leading to leakage of intracellular components.
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Fig. 7. DNA damage and binding activity of CAgNCs. (A) DNA damage analysis by agarose gel electrophoresis of genomic DNA samples extracted from A. salmonicida after CAgNCs treatment. 1: 0; 2:12.5 μg/mL, 3:25 μg/mL; 4: 50 μg/mL; 5: 75 μg/mL of CAgNCs. (B) DNA binding analysis by agarose gel electrophoresis of isolated genomic DNA (6 μg) of A. salmonicida treated with different amount CAgNCs. 1:1 Kb marker; 2: control genomic DNA; 3: 0.5 μg; 4: 1 μg; 5: 1.5 μg of CAgNCs.
Several reports have shown that bactericidal antibiotics promote the generation of ROS in E. coli which caused drug induced killing (Dwyer et al., 2012; Kohanski et al., 2008). Similarly AgNPs have been found to induce intracellular ROS levels (Su et al., 2009; You et al., 2012). On the other hand, AgNPs in the composite also can generate Ag+ and release into bacterial cell and interacts with thiol groups in proteins, resulting in inactivation of respiratory enzymes and leading to the production of ROS (Matsumura et al., 2003). Inoue et al. (2002) has described that ROS (eg. superoxide anion, hydrogen peroxide, hydroxyl radical, and singlet oxygen) contributed to the antibacterial activity against E. coli. Moreover, excessive levels of ROS can lead to damage DNA and inhibition or lowering the protein expression via oxidative stress (Marambio and Hoek, 2010). Mallick et al. (2012). Banerjee et al. (2010) documented the induction of ROS production in E. coli cell by iodinated chitosan-silver nano composite and iodine-stabilized chitosan Cu nano composite, respectively. Present study show that CAgNCs treatment generates higher ROS levels in A. salmonicida in concentration and time dependent manner. Also, results indicate the ROS induction at very low concentration (6.25 μg/μL) of CAgNCs while it maintains at higher and constant level at 50 and 75 μg/μL, respectively. The high ROS level correlates with the respective concentration of CAgNCs (50 μg/μL) at MIC level. However, ROS level was decreased (compared to 50 μg/μL) at concentration of 75 μg/μL which could be
Fig. 8. Effect of CAgNCs on ROS production in rock bream cells. Concentration dependent intracellular ROS generation was determined by the flow cytometry using H2DCFDA.
due to higher bacterial cell death. This is further supported by no any bacterial growth at 100 μg/μL which is the MBC of CAgNCs. MTT cell viability results indicated that cell viability was significantly decreased with increasing CAgNCs concentration showing 50% viable cells at 50 μg/μL which validate the MIC level of CAgNCs against A. salmonicida. It was shown that Ag+ ions prevent DNA replication and affect the structure and permeability of the cell membrane (Feng et al., 2000). Moreover, chitosan decreased the level of protein expression of S. aureus which may be due to inhibited protein synthesis or control gene expression (Tao et al., 2011; Xing et al., 2009). It reveals decreased protein expression in CAgNPs (50 μg/mL) treated A. salmonicida cells suggesting that CAgNPs could inhibit the protein synthesis. There was no clear difference of total protein under time series analysis up to 120 min and this could be due to immediate effect of CAgNCs on inactivation of protein expression (before 30 min). Furthermore, lower amount of total protein in CAgNCs treated bacteria cells could be due to loss of soluble protein as a result of membrane permeability and disrupting cell membranes as confirmed by PI and SEM analysis. Based on this, we propose that CAgNCs may regulate the inhibition of bacterial gene or protein expression in A. salmonicida and which mechanism needs to be elucidated in future. To understand the interaction between bacterial DNA and CAgNCs, the DNA binding and DNA damage assays were performed. Results confirmed that CAgNCs can strongly attach to genomic DNA of A. salmonicida in cells as well as in isolated form. It may be due to adhesion of bacterial DNA with composite through electrostatic interactions and subsequent creation of CAgNCs-DNA complex. This results is partially supported by previously study of iodine-stabilized Cu nanoparticle chitosan composite and iodinated chitosan-silver nanoparticle composite which binds the plasmid DNA of E. coli bacteria (Banerjee et al., 2010). In addition, chitosan composite binding with DNA have been reported the inhibition of mRNA synthesis of the bacteria (Dutta et al., 2009). To understand the role of CAgNCs on DNA fragmentation, we examined effect of CAgNCs on DNA degradation and it showed CAgNCs at MIC level can cause extensive cleavage of double-strand DNA that may lead to complete breakdown of genomic DNA. Thus, the complete DNA degradation appeared only at bactericidal concentration. The DNA degradation by different CAgNCs concentrations is correlated with cell death of A. salmonicida. Application of CAgNCs in bacterial control or any other therapeutic application in aquaculture, toxicity assessment is essential. Therefore, toxicity levels of CAgNCs were investigated using zebrafish (Water exposure
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Fig. 9. Rock bream cell viability detected by MTT assay. Bars represent the corresponding standard deviations (n = 3). Cell viability was assessed after treatment with different concentration of CAgNCs (6.25 μg/mL, 12.5 μg/mL, 18.75 μg/mL, 25 μg/mL and 50 μg/mL 75 μg/mL). The error bars indicate the mean ± S.D. (n = 3). Significant differences in rock bream testis cell viability were calculated with respect to untreated control (P b 0.05). The * mark represent the significantly higher inhibition.
and oral administration) and rock bream testis cells (cell viability and measuring the ROS production). Results prove that CAgNCs is not toxic to zebrafish at 12.5 mg/kg of body weight/day as a feed ingredient and rock bream testis cells up to 50 μg/μL. In conclusion, we synthesized the AgNPs embedded chitosan based CAgNCs which can effectively inhibit the fish pathogenic A. salmonicida. The cumulative effects of CAgNCs on physiological changes in cell membrane can be led to increase the cell membrane permeability and loss of cellular content in bacteria cells. Moreover, gradual release of AgNPs from chitosan matrix may promote the ROS production which can accelerate the DNA fragmentation, inhibition of protein expression and also halt the DNA repair and cellular homeostasis thereby causing for bacterial cell death. Therefore, application of significantly low toxic concentration of AgNPs in chitosan matrix in the form of composite (CAgNCs) is a much superior antibacterial agent in comparison to the use of antibiotics. Moreover, presence of chitosan may have direct effect on immune activation in fish, hence it could be considered as potential immune stimulant and vaccine adjuvant in aquaculture. Acknowledgments This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (2014R1A2A1A11054585). Dananjaya S.H.S. is supported by BK21 Plus Program, Ministry of Education, Science & Technology (MEST), Korea. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.aquaculture.2015.08.023. References Abbaszadegan, A., Ghahramani, Y., Gholami, A., Hemmateenejad, B., Dorostkar, S., Nabavizadeh, M., Sharghi, S., 2014. The effect of charge at the surface of silver nanoparticles on antimicrobial activity against gram-positive and gram-negative bacteria: a preliminary study. J. Nanomater., 720654 Akmaz, S., Adjgüzel, E.D., Yasar, M., Erguven, O., 2013. The effect of Ag content of the chitosan-silver nanoparticle composite material on the structure and antibacterial activity. Adv. Mater. Sci. Eng., 690910 Banerjee, M., Mallick, S., Paul, A., Chattopadhyay, A., Ghosh, S.S., 2010. Heightened reactive oxygen species generation in the antimicrobial activity of a three component iodinated chitosan-silver nanoparticle composite. J. Surf. Colloids 26 (8), 5901–5908. Bin, A.M., Lim, J.J., Shameli, K., Ibrahim, N.A., Tay, M.Y., 2011. Synthesis of silver nanoparticles in chitosan, gelatin and chitosan/gelatin bionanocomposites by a chemical reducing agent and their characterization. Molecules 16 (9), 7237–7248. Chen, Q., Jiang, H., Ye, H., Li, J., Huang, J., 2014. Preparation, antibacterial, and antioxidant activities of silver/chitosan composites. J. Carbohydr. Chem. 1–15. Dananjaya, S.H.S., Godahewa, G.I., Jayasooriya, R.G.P.T., Chulhong, O.H., Lee, J., De Zoysa, M., 2014. Chitosan silver nano composites (CAgNCs) as potential antibacterial agent to control Vibrio tapetis. J. Vet. Sci. Technol. 5 (5), 209. Du, W.L., Niu, S.S., Xu, Y.L., Xu, Z.R., Fan, C.L., 2009. Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions. Carbohydr. Polym. 75 (3), 385–389.
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