World J Microbiol Biotechnol DOI 10.1007/s11274-014-1674-4
ORIGINAL PAPER
Antimicrobial activity of metal based nanoparticles against microbes associated with diseases in aquaculture P. Swain • S. K. Nayak • A. Sasmal • T. Behera S. K. Barik • S. K. Swain • S. S. Mishra • A. K. Sen • J. K. Das • P. Jayasankar
•
Received: 28 December 2013 / Accepted: 20 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The emergence of diseases and mortalities in aquaculture and development of antibiotics resistance in aquatic microbes, has renewed a great interest towards alternative methods of prevention and control of diseases. Nanoparticles have enormous potential in controlling human and animal pathogens and have scope of application in aquaculture. The present investigation was carried out to find out suitable nanoparticles having antimicrobial effect against aquatic microbes. Different commercial as well as laboratory synthesized metal and metal oxide nanoparticles were screened for their antimicrobial activities against a wide range of bacterial and fungal agents including certain freshwater cyanobacteria. Among different nanoparticles, synthesized copper oxide (CuO), zinc oxide (ZnO), silver (Ag) and silver doped titanium dioxide (Ag–TiO2) showed broad spectrum antibacterial activity. On the contrary, nanoparticles like Zn and ZnO showed antifungal activity
P. Swain (&) S. K. Nayak A. Sasmal T. Behera S. K. Barik S. K. Swain S. S. Mishra Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, India e-mail: [email protected] A. K. Sen Department of Chemical Engineering and Technology, Birla Institute of Technology-Mesra, Ranchi 835215, India J. K. Das Department of Surgery, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar 751003, India P. Jayasankar Fish Genetics and Biotechnology Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, India
against fungi like Penicillium and Mucor species. Since CuO, ZnO and Ag nanoparticles showed higher antimicrobial activity, they may be explored for aquaculture use. Keywords Anti-cyanobacterial Anti-microbial Aquaculture Minimum inhibitory concentration Metal oxide Nanoparticles
Introduction The use of chemotherapeutic/antimicrobial agents is one of the most important methods of preventing and controlling infectious agents during past few decades. However, routine use of these agents leads to several adverse consequences. The rapid development of antibiotic resistance has emerged as one of the serious problems affecting all forms of life and aquatic species are no exception (Aarestrup 2005; Rice 2009). Over the years, scientific reports have been conclusive with respect to the health risk associated with indiscriminate use of antibiotics in fishes and other aquatic organisms. The indiscriminate use of antibiotics adversely affects the living systems, environment (Hektoen et al. 1995; Cabello 2006) and often leads to the development and spread of highly drug resistance pattern among pathogens. Several pathogens such as Aeromonas, Pseudomonas, Edwardsiella, Listeria, Staphylococcus, Vibrio species etc. are reported to be pathogens for both aquatic species and humans which further enhance the possibility of horizontal gene transfer (Novotny et al. 2004; Tenover 2006). Therefore, there is a need to search alternative approch to control pathogens. Diseases are the major stumbling block towards sustainable growth of the aquaculture. Fish are threatened by many pathogens, especially viruses and bacteria, often with
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serious consequences (Smith 2012; Romero et al. 2012; Leung and Bates 2013). Due to the increase in disease outbreak and the development of microbial resistance, there is an urgent need to prevent infectious agents and other harmful microorganisms without propagating antibiotic resistance pattern (Kemper 2008; Romero et al. 2012). One of the possibilities is to use nanoparticles as antimicrobial drug in aquaculture but their potential use for disease control is not fully explored yet. Recently, much attention has been paid towards the use of nanoparticles as an alternative to antibiotics due to their distinct advantages over conventional antimicrobial agents (Gunalan et al. 2012). The unique, unusual and interesting physical, chemical, and biological properties of nanometersized materials have recently attracted a great deal of interest from the scientific community. Nanoparticles with one dimension of 100 nm or less in size are now being increasingly utilised for medical applications and is of great interest as an alternative approach to control infectious agents. Among different types of nanoparticles, metals and metal oxides have elicited a great deal of interest due to their broadspectrum of antimicrobial activity, durability, high resistance, selectivity and specificity (Singh et al. 2012). Metal based nanoparticles are demonstrated to be excellent antimicrobial agent and exhibit broad spectrum antimicrobial activity against bacteria, fungi and viruses (Ren et al. 2009; Ali et al. 2011; Galdiero et al. 2011; Nasrollahi et al. 2011; Seil and Webster 2012). Therefore, the aim of the present study was to investigate the antimicrobial activity of different metal nanoparticles (both commercial and laboratory synthesized) as potential antimicrobial agent(s) for use in aquaculture practices.
Materials and methods Microorganisms Different bacterial and fungal isolates were used in the present investigation. Bacterial isolates such as Aeromonas hydrophila (FHM/AhV/2000), Edwardsiella tarda (FHM/Et/2000), Pseudomonas aeruginosa (FHM/Pa/2000), Flavobacterium branchiophilum (FHM/Fbr/2002) and other bacteria like Vibrio species (FHM/V/2000), Staphylococcus aureus (FHM/ Sa/2001), Bacillus cereus (FHM/Bc/2004) and Citrobacter species (FHM/C/2004) and four fungal isolates namely Aspergillus (FHMA/2000), Penicillium (FHMP/2000), Fusarium (FHMF/2004) and Mucor species (FHMM/2005) were used in this study. They were isolated from various disease affected freshwater fishes, characterized and maintained at Fish Health Management Division, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Odisha, India, as repository. Three cyanobacterial isolates belonging
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Table 1 Different nanoparticles used to screen antimicrobial activities in the study Sl. no.
Nanoparticles
Symbol/ formulae
Form
Size (in nm)
Commercial nanoparticles 1.
Zinc
Zn
Powder
2. 3.
Zinc oxide Zinc oxide
ZnO (C) ZnO (D)
Powder Dispersion
\50 \100 \35
4.
Copper oxide
CuO (C)
Powder
\40
5.
Iron oxide
Fe2O3
Powder
\50
6.
Iron oxide
Fe2O3
Dispersion
\30
7.
Aluminium oxide
Al2O3
Dispersion
\50
8.
Silver
Ag (D)
Dispersion
10
9.
Silver
Ag (C)
Powder
100
Synthesized (chemical and green method) nanoparticles 10.
Zinc oxide (chemical)
ZnO (S)
Powder
122.4
11.
Copper oxide (chemical)
CuO (S)
Powder
93
12.
Silver doped Titanium dioxide (chemical) Silver (green)
Ag– TiO2 Ag (S)
Powder
91.82
Suspension
58.7
13.
to Oscillatoria species (one strain) and Anabaena species (two strains), were obtained from the Department of Microbiology, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India and used in this study. Nanoparticles Both commercially available and laboratory synthesized nanaopaticles were used in the present study. Zinc (Zn); zinc oxide (ZnO); copper oxide (CuO); iron oxide (Fe2O3 powder/dispersion); aluminium oxide (Al2O3) and silver (Ag, powder/dispersion) were procured from Sigma USA. Silver (Ag), zinc oxide (ZnO); copper oxide (CuO) and silver doped titanium dioxide (Ag–TiO2) nanoparticles were synthesized in the laboratory as described below. Detailed information regarding the size and form of various nanoparticles is given in Table 1. Ag nanoparticles The green synthesis of Ag nanoparticles from AgNO3 using locally available lemon (Citrus limon) was prepared as per the procedure of Prathna et al. (2011) with slight modifications. Briefly, lemon juice was mechanically extracted from fresh lemons and then strained through a fine nylon mesh followed by centrifugation at 10,0009 rpm for 10 min at 4 °C. After centrifugation, supernatant was collected and processed for synthesis of Ag nanoparticles. The Ag nanoparticles were prepared by adding lemon extract to varying molar concentration of AgNO3 (10-2 M to 10-4 M) at different ratio of 2:3, 1:1, 3:2 and 4:1.
World J Microbiol Biotechnol
Finally, solutions containing different combinations of lemon extract and AgNO3 were incubated at 60 °C in a water bath for 3 h during which change in colour of the solution from pale yellow to reddish brown took place indicating the formation of silver nanoparticles. CuO nanoparticles CuO nanoparticles were prepared as per the procedure of Lanje et al. (2010). Briefly, aqueous solution of copper acetate (0.02 M) was prepared and then 1 ml glacial acetic acid was added to the aqueous copper acetate solution. The solution was then heated to 100 °C with constant stirring. About 0.4 g of NaOH was added to above heated solution till pH reached 6–7. The formation of CuO nanoparticles was indicated by the immediate color change of the solution from blue to black. The solution was centrifuged at 15,0009 rpm for 30 min followed by three times washing with de-ionized water and finally air dried for 24 h. The dried CuO nanoparticles powder was collected in clean sterilized vials.
drop of each sample was placed on clean glass slide. After drying, the samples were sputter coated with a thin layer of 60 % gold and 40 % palladium for 30 s with 45 mA current. The morphological feature of the synthesized nanoparticles was then observed on the SEM (JEOL JSMT220A, MA) using an accelerating voltage of 5–7 kV. The average particle size of CuO, ZnO, Ag–TiO2 and Ag (only for lemon and AgNO3 ratio of 3:2) nanoparticles were determined by DLS using a laser diffraction method with a multiple scattering technique. The synthesized nanoparticles, dispersed in distilled water was first sonicated at 40 Hz for 30 min and then the samples were analyzed in a computer controlled particle size analyzer (ZETA Sizers Nano ZS, Malvern Instruments USA). Similarly, the preliminary characterization/particle size of Ag nanoparticles was determined by measuring the UV– visible spectra of the diluted synthesized solutions (1:20 in deionized water) from 200 to 600 nm range. Determination of antimicrobial activity by the disc diffusion assay
ZnO nanoparticles ZnO nanoparticles were prepared by a wet chemical method using aqueous solution of ZnCl2 (0.5 M) and NaOH (1 M) precursors as per the method of Moghaddam et al. (2009) with slight modifications. Briefly, NaOH (1 M) solution was first heated to 50 °C for 5 min and then ZnCl2 was added drop wise under high magnetic stirring. After addition of ZnCl2, the solution was sealed and stirred for further 10 min at 50 °C till white precipitate formed. ZnO nanoparticles containing pellet was washed three times with distilled water followed by twice washing with ethanol. Finally, ZnO nanoparticles were collected after drying the pellet at 50 °C. Ag doped TiO2 Ag doped TiO2 (Ag–TiO2) nanoparticles were prepared as per Saha et al. (2012) method with slight modifications. Briefly, AgNO3 was dissolved in distilled water (1.2 wt%) followed by addition of TiO2 powder (55 wt%). After addition, a paste was formed which was then dried in hot air oven at 100 °C for 2 h. This was followed by further heating in muffle furnace at 400 °C for 4 h and finally at 600 °C for 1 h. The powder was grinded in a ball mill for 2 h to decrease the particle size. Characterization of synthesized nanoparticles The morphological features of CuO, ZnO and Ag–TiO2 nanoparticles were further characterized by scanning electron microscopy (SEM). First of all, the synthesized nanoparticles were sonicated with distilled water and then one
Antimicrobial activities of nanoparticles were determined using different aquatic microbes following disc diffusion assays using the protocol of Bauer et al. (1966). Overnight grown bacterial cultures in nutrient broth (Hi-Media, Mumbai, India) (OD540 = 0.5) were individually lawn cultured on nutrient agar (Hi-Media, Mumbai, India) plates. Sterile filter paper discs containing 5 mg of nanoparticles were placed above the culture and then the plates were incubated at 30 °C for 120 h after which, the zone of inhibition were recorded. Based on the findings of inhibition zone after incubation (conducted in triplicate), the results were interpreted either as positive or negative. The nanoparticles showing inhibitory activities were further evaluated to determine minimum inhibitory concentration (MIC). Determination of minimum inhibitory concentration (MIC) The nanoparticles showing positive inhibitory activities in the preliminary disc diffusion assays were further processed to find out the MIC against different bacteria. The MIC values were calculated as per standard microbiological tube dilution method. Briefly, nutrient broth was added with individual nanoparticles ranging from 0 to 5 mg/ml (5.0, 2.5, 1.25, 1.0, 0.5, 0.62, 0.31, 0.25, 0.15, 0.1, 0.078, 0.062, 0.031, 0.025, 0.015, 0.01, 0.005, 0.007 and 0.001 mg/ml). Then 100 ll of target pathogen (at 106 CFU/ml) were added into the nanoparticles containing nutrient broth and then incubated at 30 °C overnight. After that, bacterial growth was monitored by measuring OD at 600 nm. An aliquot was streaked on nutrient agar plates. The MIC values were
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determined as the lowest concentration of nanoparticles showing complete inhibition of bacterial growth after overnight incubation. Anti-cyanobacterial assay Nanoparticles at varying concentrations (as specified for MIC value determination in antibacterial assay) were added to 10 ml of Chu-10 medium (Hi-Media, India) before inoculating with freshly grown cyanobacteria cultures. The treated cultures were incubated under diffuse florescent tube light illumination with a day/night rate of 16/8 h with gentle shaking thrice a day to avoid clumping. The MIC values were determined as the lowest concentration of nanoparticles showing complete inhibition of cyanobacteria growth after 3 weeks of culture. Effect of nanoparticles on bacterial growth Effect of laboratory synthesized CuO, ZnO and Ag nanoparticles on the growth of A. hydrophila was studied by using standard microbial methods. Briefly varying concentrations of synthesized nanoparticles (0, 0.31, 0.62, 1.25, 2.5 and 5.0 mg/ml) were separately incubated with A. hydrophila (106 to 1011 CFU/ml) overnight at 30 °C. After that the OD was recorded at 600 nm. Similar type of kinetics study was also conducted against with A. hydrophila (106 to 1011 CFU/ml) with synthesized Ag nanoparticles at various dilutions (Neat, 1/2, 1/4, 1/8, 1/16, 1/25, 1/50, 1/100) of lemon extract and AgNO3 as controls. Anti-fungal assay The antifungal activity of different nanoparticles was determined as per the disc diffusion method. Briefly, a loop full of fungal mycelia was inoculated at the center of a Sabouraud dextrose agar (SDA, Hi-Media, India) plates and then sterile filter paper discs containing individual nanoparticles suspension at varying concentrations were placed in the periphery. The concentrations used were same as specified for MIC value determination in antibacterial assay. The plates were incubated at room temperature for 7 days and were monitored for inhibitory zone. The MIC values were determined as the lowest concentration of nanoparticles showing complete inhibition of fungal growth after 7 days of incubation.
Fig. 1 The SEM micrographs showing the surface morphology of synthesized a CuO, b ZnO and c Ag–TiO2
Results Characterization of synthesized nanoparticles The surface morphology of the laboratory synthesized CuO, ZnO and Ag–TiO2 nanoparticles are shown in the
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Fig. 1a–c. SEM micrographs of the CuO showed free and spherical forms of nanoparticles. On the contrary, ZnO nanoparticles were found to be aggregated, forming a microstructure, while Ag–TiO2 nanoparticles were found to be densely packed spherical forms.
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Fig. 3 The UV spectra of green synthesized Ag nanoparticles using different ratio of AgNO3 and Lemon
mean percentage of particles were maximum (32.9 %) with peak at 93 nm for synthesized CuO while 83.8 % of ZnO were 122.4 nm in size. Ag–TiO2 also revealed a broad size distribution with particles in the size range of 43.8 to 190.1 nm with a mean percentage of particles maximum (15 %) at a peak value of 91.82 nm. The formation of silver nanoparticles obtained from varying the mixing ratios between AgNO3 and lemon extract were also confirmed by UV–visible spectroscopy (Fig. 3). Among four different combinations of lemon extract and AgNO3 used to prepare Ag nanoparticles, the peak was obtained at 460 nm in 4:1 ratio, 420 nm in 3:2 ratio, 439 nm in 1:1 ratio and 441 nm in 2:3 ratio of lemon extract and AgNO3. The particles size of the Ag nanoparticles (of 3:2 ratio) as determined from DLS study ranged from 43 to 140 nm with peak at 58.7 nm. Anti-bacterial activity
Fig. 2 The average particle size of various laboratory synthesized nanoparticles as determined by DLS. a CuO, b ZnO, c Ag–TiO2 and d Ag (Lemon 3: 2 AgNO3)
The size distribution pattern of the laboratory synthesized (CuO, ZnO, Ag–TiO2 and Ag nanoparticles) as determined by DLS study is shown in Fig. 2a–d. The particle distribution curve was broad for CuO whereas that of ZnO were more uniform with a narrow range of distribution. The particle size of CuO and ZnO ranged from 58.77 to 141.8 and 105.7 to 122.4 nm, respectively. The
Different nanoparticles exhibited antibacterial activities against various bacterial isolates by disc diffusion assay (Fig. 4a–d). Among different nanoparticles, commercial nanoparticles like ZnO and CuO were found to inhibit the bacteria but Zn, Ag, Fe2O3, and Al2O3 nanoparticles failed to inhibit any of the tested isolates. But in case of commercial ZnO nanoparticle treatment, over growth of B. cereus, P. aeruginosa and Vibrio species, was recorded upon prolonged incubation upto 120 h. Likewise, the chemical synthesized nanoparticles, ZnO and CuO were found to inhibit all bacterial isolates irrespective of incubation period. However, chemical synthesized Ag–TiO2 nanoparticles only inhibited A. hydrophila, E. tarda, F. branchiophilum, S. aureus and Citrobacter species but on
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Fig. 4 Disc diffusion assays showing zone of inhibition exhibited by various nanoparticles against different bacterial isolates. a B. cereus; b P. aeruginosa; c E. tarda; d A. hydrophila. Numerical numbers in
the individual disc of individual plate (a–d) represent different types of nanoparticles 1 CuO(S); 2 CuO(C); 3 ZnO(S); 4 ZnO(D); 5 Ag; 6 Zn 7 Al2O3; 8 Fe2O3
prolonged incubation upto 120 h, overgrowth was recorded (Table 2). On the other hand, Ag nanoparticles prepared by different combinations of lemon and AgNO3 exhibited antibacterial activities. With lemon extract as a control treatment, highest zone of inhibition against E. tarda and S. aureus was recorded in lemon extract diluted with water at 4:1 ratio followed by 3:2, 1:1 and 2:3 ratios. However, the zone of inhibition was highest against P. aeruginosa at 2:3 dilution while no variation in the zone size could be recorded at any dilutions of lemon extract against A. hydrophila. Similarly, in AgNO3 control group, AgNO3 at 10-3 M and 10-4 M did not exhibit any antibacterial activity since no zone of inhibition against any of the tested bacteria could be observed. However, 10-2 M AgNO3 exhibited highest inhibitory zone against all bacteria. On the contrary, highest zone of inhibition was recorded in the
Ag nanoparticles prepared from lemon 10-2 M and AgNO3 at 3:2 ratio (Table 3).
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MIC values determination The MIC values of various commercial and laboratory synthesized nanoparticles against different bacterial pathogens is given in Table 4. The MIC values of commercial CuO and laboratory synthetized ZnO nanoparticles against most of the pathogens were found to be more than 5 mg/ml. On the contrary, chemically synthesized nanoparticles, CuO nanoparticles showed lower MIC values against most of the pathogens as compared to that of ZnO and Ag–TiO2 nanoparticles. The MIC values were found to be more than 5 mg/ ml for ZnO and Ag–TiO2 nanoparticles . The MIC values of laboratory synthesized Ag nanoparticles against different bacterial isolates are given in
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Table 5. The least MIC values against various bacteria were recorded when Ag nanoparticles were prepared at 3:2 dilution of Lemon: AgNO3 while highest MIC values were recorded when Ag nanoparticles were prepared at 2:3 dilution of Lemon extract: AgNO3. Anti-cyanobacterial activity Nanoparticles were found to inhibit the growth of Oscillatoria and Anabaena species (Table 6) but the minimum inhibitory dose with regard to nanoparticles varied. Among the nanoparticles, Fe2O3 was found to be most effective nanoparticles with least MIC values of 0.015 and
Table 2 Zone of inhibition exhibited by various nanoparticles against different bacterial isolates Sr. no
Nanoparticles
Pathogens 1
2
3
4
5
6
7
8
Commercial nanoparticles 1.
Zinc (Zn)
-
-
-
-
-
-
-
-
2.
Zinc oxide (ZnO) (C)
?
?
?
?
?
-
-
-
3. 4.
Zinc oxide (ZnO) (D) Copper oxide (CuO) (C)
? ?
? ?
? ?
? ?
? ?
? ?
? ?
? ?
5.
Iron oxide (Fe2O3)
-
-
-
-
-
-
-
-
6.
Iron oxide (Fe2O3) (D)
-
-
-
-
-
-
-
-
7.
Aluminium oxide (Al2O3)
-
-
-
-
-
-
-
-
8.
Silver (Ag) (D)
-
-
-
-
-
-
-
-
9.
Silver (Ag) (C)
-
-
-
-
-
-
-
-
Synthesized nanoparticles
0.062 mg/ml against two Anabaena species while that of Oscillatoria species was 0.031 mg/ml. Apart from Fe2O3, CuO(S), Ag(S) and ZnO(D) were also effective with low MIC values against all the three cyanobacteria. However, among the three isolates of cyanobacteria, Oscillatoria showed higher resistance towards nanoparticles and the MIC values were found to be higher than 5 mg/ml against ZnO(C), Ag(C) and Al2O3. Both the Anabaena species were completely inhibited by CuO(C), Ag–TiO2, and Al2O3 with MIC values of 0.062, 0.078 and 0.5 mg/ml respectively. Effect of nanoparticles on bacterial growth Effect of different concentration of laboratory synthesized CuO nanoparticles on A. hydrophila is shown in Fig. 5a. CuO nanoparticles were found to reduce the growth of A. hydrophila in a concentration dependent manner. Complete inhibition of A. hydrophila upto 1010 CFU/ml was recorded for varying concentration of CuO nanoparticles ranging from 0.31 to 5 mg/ml. However, growth of A. hydrophila at 1011 CFU/ml was recorded even at treatment of CuO nanoparticles at 5 mg/ml. But, less growth was recorded as Table 4 MIC values of various commercial and laboratory synthesized nanoparticles against different bacterial agents Sr. no
Bacteria
MIC values (mg/ml)
1
Aeromonas hydrophila
ZnO (D)
ZnO CuO (S) (C)
CuO (S)
Ag– TiO2
0.25
2.5
1.2
0.31
1.2
10.
Zinc oxide (ZnO) (S)
?
?
?
?
?
?
?
?
2
Edwardsiella tarda
0.62
0.31
0.62
2.5
0.25
11.
Copper oxide (CuO) (S)
?
?
?
?
?
?
?
?
3
Bacillus cereus
[5
[5
[5
1.2
[5
12.
Silver–Titanium dioxide (Ag–TiO2)
?
?
?
?
?
-
-
-
4
Citrobacter species
1.2
0.62
2.5
1.2
0.25
5
1.2
[5
[5
2.5
0.25
Ag (S)
?
Flavobacterium branchiophilum
0.31
13.
?
?
?
?
?
?
?
1: A. hydrophila 2: Citrobacter species 3: E. tarda 4: F. branchiophilum 5: S. aureus 6: Vibrio species 7: B. cereus 8: P. aeruginosa
6
Staphylococcus aureus
[5
1.2
[5
0.16
7
Vibrio species
[5
[5
[5
0.31 [5
? Inhibition, - No inhibition
8
Pseudomonas aeruginosa
[5
[5
[5
0.16 [5
Table 3 Zone of inhibition (in mm) against different bacterial agents exhibited by green synthesized Ag nanoparticles
Lemon: AgNO3 ratio
A. hydrophila
E. tarda
S. aureus
P. aeruginosa
4:1
3:2
1:1
2:3
4:1
3:2
1:1
2:3
4:1
3:2
1:1
2:3
4:1
3:2
1:1
2:3
10-2
18
21
18
20
10
16
11
16
20
25
21
24
16
16
17
18
10-3
9
9
9
9
10
10
10
10
10
10
10
9
9
8
8
9
10-4
9
9
9
9
11
10
9
9
10
10
9
9
8
8
8
9
AgNO3 (10-2)
12
20
18
18
10
14
13
17
18
23
21
24
12
14
16
17
AgNO3 (10-3)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
AgNO3 (10-4)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Lemon extract
8
8
8
8
11
10
9
9
10
10
9
9
8
7
8
9
Control groups
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the fungi were found to be 2.5 mg/ml. Similarly, both forms of ZnO nanoparticles i.e., ZnO(S) and ZnO(D) were also found to inhibit Penicillium and Mucor species with MIC value of 2.5 and 5.0 mg/ml, respectively. On the contrary, Zn and ZnO nanoparticles, the MIC values were higher than 5.0 mg/ml for Aspergillus and Fusarium species. Apart from these nanoparticles, others nanoparticles like CuO(S), CuO(C), Fe2O3, Al2O3 Ag(C) and Ag(S) did not exhibit antifungal activity up to 5 mg/ml against any of the tested fungi (Table 7).
compared to control group. Similar types of inhibitory pattern against A. hydrophila was also recorded for ZnO nanoparticles (Fig. 5b). On the other hand, Ag nanoparticles synthesized from AgNO3 using lemon extract irrespective of dilution completely inhibited the growth of A. hydrophila up to 1010 CFU/ml while growth was recorded in the group treated with A. hydrophila (1011 CFU/ml) Ag nanoparticles at 1/100 and 1/50 dilution. On the contrary, complete inhibition of A. hydrophila growth was recorded at 1/2 dilution of lemon extract while that of AgNO3 (10-2 M) was observed at 1/8 dilution. At 1/50 dilution, lemon and AgNO3 (10-2 M) showed very low antibacterial activity with the growth pattern similar to that of control (Fig. 5c).
Discussion The field of nanotechnology is rapidly expanding and used in various areas, such as health management, food and feed, environmental aspects and agricultural practices (Roszek et al. 2005; Bouwmeester et al. 2007; Kiruba Daniel et al. 2013). Over the years, nanoparticles have achieved unprecedented success in all areas of their applications for the benefit of society. With unique
Anti-fungal activity Among all the tested nanoparticles, Zn and ZnO showed antifugal activity against Penicillium and Mucor species (Fig. 6a, b). Zn nanoparticles, were found to inhibit Penicillium and Mucor species and the MIC values against both Table 5 MIC values (in mg/ml) of green synthesized Ag nanoparticles against different bacterial agents
Table 6 MIC values (in mg/ ml) of various commercial and laboratory synthesized nanoparticles against different cyanobacteria
Table 7 MIC values (in mg/ ml) of various commercial and laboratory synthesized nanoparticles against different fungi
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Sr. no
Bacteria
Minimum inhibitory concentration (mg/ml) Lemon:AgNO3 (1:1)
Lemon:AgNO3 (2:3)
Lemon:AgNO3 (3:2)
1
Aeromonas hydrophila
2.7
6.4
0.21
2
Edwardsiella tarda
2.7
6.4
0.21
3
Bacillus cereus
2.7
6.4
0.21
4
Citrobacter species
2.7
6.4
0.21
5
Flavobacterium branchiophilum
2.7
6.4
0.21
6
Staphylococcus aureus
2.7
1.6
0.21
7
Vibrio species
1.3
1.6
0.1
8
Pseudomonas aeruginosa
1.3
1.6
0.1
Sr. no
Species
Minimum inhibitory concentration (mg/ml) Fe2O3
Zn
ZnO (D)
ZnO (S)
CuO (C)
CuO (S)
Ag (C)
Ag (S)
Al2O3
Ag–TiO2
1
Anabaena
0.015
0.12
0.078
0.25
0.062
0.062
[5
0.062
0.5
0.078
2
Anabaena
0.062
0.25
0.078
0.12
0.062
0.031
[5
0.062
0.5
0.078
3
Oscillatoria
0.031
0.25
0.015
[5
0.12
0.078
[5
0.078
[5
0.25
Sr. no
Species
Minimum inhibitory concentration (mg/ml) Fe2O3
Zn
ZnO (D)
ZnO (S)
CuO (C)
CuO (S)
Ag (C)
Ag (S)
Al2O3
Ag–TiO2
2.5
2.5
2.5
[5
[5
[5
[5
[5
[5
1
Penicillium
[5
2
Mucor
[5
2.5
5
5
[5
[5
[5
[5
[5
[5
3
Fusarium
[5
[5
[5
[5
[5
[5
[5
[5
[5
[5
4
Aspergillus
[5
[5
[5
[5
[5
[5
[5
[5
[5
[5
World J Microbiol Biotechnol
Fig. 5 Effects of synthesized nanoparticles on the growth of A. hydrophila at 106 to 1011 CFU/ml. (a CuO; b ZnO; c: Ag)
properties like high surface volume ratio, ability to withstand intensive processing conditions, depressed melting temperature, conductivity, magnetism and light absorption, nanoparticles have gained tremendous advantages as compared to their large sized counterparts (Feynman 1991). Recently, metals and/or their oxide nanoparticles by virtue of their physico-chemical properties have emerged as promising antimicrobial agents. These nanoparticles can adopt various mechanisms like inhibition of the synthesis of functional biomolecules or impeding normal cellular activities to kill pathogens (Rai and Bai 2011).
Fishes are susceptible to a wide variety of microbial agents among which bacterial diseases are most prevalent causing severe economic losses throughout world. Numerous species of bacteria are capable of causing diseases in fish and the emergence of antimicrobial resistance by pathogenic bacteria is also a major concern. Similarly, fungal infections are common, especially under stress conditions and poor water quality (Verma 2008). Furthermore, many times cyanobacterial blooms are affecting aquaculture throughout world and cyanotoxin like microcystins is highly toxic to fish at micrograms level (Carbis et al. 1996; Fischer et al. 2000; Malbrouck and Kestemont 2006; Ernst 2008). Therefore, as an alternate approach, we have evaluated a battery of nanoparticles both commercial and laboratory synthesized, for their antimicrobial activity against a broad range of microbial agents. We have synthesized the Ag–TiO2, CuO, ZnO and Ag nanoparticles in laboratory. The mean percentage of particles were maximum (32.9 %) with peak at 93 nm for synthesized CuO, 83.8 % of ZnO nanoparticles were of 113 nm in size and Ag–TiO2 revealed a maximum (15 %) at a peak value of 91.82 nm. Similarly, we also synthesized Ag nanoparticles using lemon extract as a suitable reducing agent. Ag nanoparticles synthesized in 4 combinations of lemon extract and AgNO3 showed peak at range from 420 to 460 nm and was in accordance to earlier report (Prathna et al. 2011). The UV-visible spectra of indicated size of Ag nanoparticles using lemon extract and AgNO3 (10-2 M) in the ratio 3:2 to vary in 50.78 to 78.82 nm which was also corroborated from the DLS data. Among various nanoparticles, ZnO, CuO, Ag and Ag– TiO2 demonstrated antimicrobial activities. On the contrary, commercial Ag, Fe2O3 and Al2O3 showed no antibacterial activity upto a concentration of 5 mg/ml. In this study, we have also demonstrated antibacterial activity of CuO nanoparticles against all the tested bacterial agents. Similar types of antibacterial activity of CuO nanoparticles have been demonstrated against different bacterial species by earlier researchers (Baek and An 2011; Azam et al. 2012; Abboud et al. 2013). However, among the CuO nanoparticles, the particle size of commercial CuO was \40 nm as compared to higher size (B93 nm) of the laboratory synthesized CuO but synthesized CuO nanoparticles exhibited better antimicrobial activities with least MIC values than commercial CuO nanoparticles. The present findings differ from the earlier findings of Azam et al. (2012) who had reported the size dependent antimicrobial activity of CuO nanoparticles. They have also recorded CuO nanoparticles of 20 (±1.24) nm size to inhibit significantly various pathogens as compared to the higher sized CuO nanoparticles. Such difference in the antibacterial activity might be attributed to the difference in dissolving properties and subsequent aggregation of
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Fig. 6 Antifungal activity of Zn and ZnO nanoparticles at different concentration against a Penicillium and b Mucor species after 7 days of inoculation. Numerical numbers in the individual disc of individual
plate (6a, b) indicate the concentration of nanoparticles: 1 Zn (5.0 mg/ml); 2 Zn (2.5 mg/ml); 3 ZnO(S) (5.0 mg/ml); 4 ZnO (S) (2.5 mg/ml); 5 ZnO(D) (2.5 mg/ml); 6 ZnO (D) (5.0 mg/ml)
commercial CuO nanoparticles in the aqueous environment could have created hindrance in direct interaction between particles and microorganisms (Wu et al. 2010). Similarly, ZnO nanoparticles have a wide range of applications in various fields due to their unique and superior physical and chemical properties compared with bulk ZnO. Earlier studies showed size dependent inhibitory activity of nanoparticles including ZnO (Jones et al. 2008; Nair et al. 2009; Raghupathi et al. 2011). ZnO nanoparticles of 13 nm size were found to inhibit S. aureus growth at 0.081 mg/ml (Reddy et al. 2007) while ZnO nanoparticles of size 60 nm at 0.4 mg/ml only reduced 50 % of S. aureus (Jones et al. 2008). Herein, we have also found higher MIC value of 1.2 mg/ml against S. aureus by ZnO nanoparticles of 122.4 nm size. This could be attributed to better solubility of synthesized ZnO nanoparticles in aqueous medium. However, we failed to record inhibitory activity of commercial ZnO of \100 nm against bacteria like Vibrio species, B. cereus and P. aeruginosa where the MIC values were found to be more than 5 mg/ml. Such difference could be attributed to the species specific differences. The antimicrobial effect of Ag nanoparticles has already been demonstrated in several studies (Kvitek et al. 2008; Dror-Ehre et al. 2009; Panacek et al. 2009; Kim et al. 2011). We have synthesized Ag nanoparticles using lemon extract as a suitable reducing agent and demonstrated excellent antibacterial activities against a broad range of pathogens with very low MIC values. However, commercial Ag did not show antibacterial activity and the MIC values were more than 5 mg/ml against all bacterial isolates which might be due to their agglomerating nature during commercial preparations as compared to green synthesized method. However, the Ag synthesized using
lemon failed to exhibit any anti-fungal activities. Ag–TiO2 nanoparticles exhibited antibacterial activity against specific bacteria. The antimicrobial activity of Ag–TiO2 may be attributed to the facts that the small particle size of Ag? could enhance its solubility and the presence of extracellular Ag? could interfere with the intracellular Ca? metabolism and cause cellular damage (Korbekandi and Iravani 2012). Further TiO2 might be also responsible in enhancing the activity via photocatalysis or generation of reactive oxygen species (Wong et al. 2010). In this study, we have also included four fungal species that are associated with Aquaculture. Penicillium and Mucor species were sensitive to Zn and ZnO. Limited information’s are available on the antifungal activity of ZnO nanoparticles (Chitra and Annadurai 2013). However, available reports indicate the antifungal activities of ZnO nanoparticles against different fungi such as Penicillium, Aspergillus, Fusarium etc. from different sources (He et al. 2011; Chitra and Annadurai 2013; Dimkpa et al. 2013). He et al. (2011) also observed inhibitory activity of ZnO of 70 (±15) nm at minimum of 3 mmol/l (&0.244 mg/ml) against Penicillium expansum. Gunalan et al. (2012) demonstrated that higher sized ZnO (40 nm) to execute enhanced antimicrobial activity against bacteria and fungus when compared to ZnO of lower particle size (25 nm). Herein, ZnO nanoparticles were found to be effective against Penicillium and Mucor species. But the findings are contradictory to that of Chitra and Annadurai (2013) who demonstrated the antifungal activity of ZnO nanoparticles (100–146 nm) against Aspergillus niger with a maximum inhibition concentration of 0.4 mg. So there may be species specific inhibitory activities exerted by ZnO. Also effectiveness of nanoparticles increases with increasing particle
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dose, treatment time and synthesis method. Additionally variation in the particle size and surface area to volume ratio of the synthesized ZnO nanoparticles are possibly contributed to better antimicrobial activity. ZnO was found to inhibit the growth of Penicillium and Mucor species at 2.5 and 5 mg/ml, respectively. The inhibitory activity of ZnO nanoparticles against different fungi is in agreement with earlier reports (Lipovsky et al. 2011). However, Aspergillus and Fusarium species were resistant upto 5 mg/ml. The antifungal activity of ZnO may be due to suppression of extracellular enzymes and metabolites that helps its survival when exposed to stress as demonstrated by fungi like Aspergillus fumigatus, Trichoderma reesei (Bhainsa and D’ Souza 2006; Vahabi et al. 2011). While a bulk of informations are already available on the effect of nanoparticles on bacteria and fungi, till date only a few reports are available on the effect of nanoparticles on unicellular green algae (Franklin et al. 2007; Dash et al. 2012). Among different cyanobacterial species, Anabaena and Oscillatoria species are the two common microcystins producing toxic cyanobacteria found mostly in freshwater system. Here, we have demonstrated that nanoparticles like Fe2O3, CuO(S), Ag(S) and ZnO(D) to be very effective against both the species of Anabaena and Oscillatoria species. It is therefore, summarized from the present study that nanoparticles, especially synthesized CuO, ZnO and Ag have excellent antimicrobial activity and could be further explored as substitute of antibiotics in aquaculture use. However, there is also a need to understand the detailed antimicrobial mechanism(s) of various metal nanoparticles against other microbes dwell in the aquaculture system and an in-depth study is further required for accessing the risks associated with them before their application in aquaculture. Acknowledgments The authors are thankful to Indian Council of Agricultural Research (ICAR), New Delhi for funding the project under the National Fellow Scheme. The authors are also thankful to the Director of Central Institute of Freshwater Aquaculture, Kausalyaganga, Odisha, India for providing necessary facility to carry out the present work. Conflict of interest
None.
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Ali DM, Thajuddin N, Jeganathan K, Gunasekaran M (2011) Plant extract mediated synthesis of silver and gold nanoparticles and its antibacterial activity against clinically isolated pathogens. Colloids Surf B 85(2):360–365 Azam A, Ahmed AS, Oves M, Khan MS, Habib SS, Memic A (2012) Antimicrobial activity of metal oxide nanoparticles against Gram-positive and Gram-negative bacteria: a comparative study. Int J Nanomed 7:6003 Baek Y, An Y (2011) Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis and Streptococcus aureus. Sci Total Environ 409:1603–1608 Bauer AW, Kirby WMM, Sherris JCT, Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 45(4):493 Bhainsa KC, D’ Souza SF (2006) Extracellular biosynthesis of silver nanoparticles using the fungus Aspergillus fumigatus. Colloids Surf B 47(2):160–164 Bouwmeester H, Dekkers S, Noordam M, Hagens W, Bulder A, De Heer C, Ten Voorde S, Wijnhoven S, Sips A (2007) Health impact of nanotechnologies in food production. Wageningen UR, RIKILT. Report 2007.014, published by RIKILT—Institute of Food Safety, Wageningen UR and National Institute of Public Health & the Environment—Center for Substances and Integrated Risk Assessment. Accessed at. http://edepot.wur.nl/ 120669; pp 1–91 Cabello FC (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 8(7):1137–1144 Carbis CR, Mitchell GF, Anderson JW, McCauley I (1996) The effects of microcystins on the serum biochemistry of carp, Cyprinus carpio L, when the toxins are administered by gavage, immersion and intraperitoneal routes. J Fish Dis 19:151–159 Chitra K, Annadurai G (2013) Antimicrobial activity of wet chemically engineered spherical shaped ZnO nanoparticles on food borne pathogen. Int Food Res J 20(1):59–64 Dash A, Singh AP, Chaudhary BR, Singh SK, Dash D (2012) Effect of silver nanoparticles on growth of eukaryotic green algae. Nano-Micro Lett 4(3):158–165 Dimkpa CO, McLean JE, Britt DW, Anderson AJ (2013) Antifungal activity of ZnO nanoparticles and their interactive effect with a biocontrol bacterium on growth antagonism of the plant pathogen Fusarium graminearum. Biometals 26(6):913–924 Dror-Ehre A, Mamane H, Belenkova T, Markovich G, Adin A (2009) Silver nanoparticle—E. coli colloidal interaction in water and effect on E. coli survival. J Colloid Interface Sci 339(2):521–526 Ernst B (2008) Investigations of the impact of toxic cyanobacteria on fish—as exemplified by the coregonids in lake Ammersee. Dissertation, University of Kostanz Feynman R (1991) There’s plenty of room at the bottom. Science 254:1300–1301 Fischer WJ, Hitzfeld BC, Tencalla F, Eriksson JE, Mikhailov A, Dietrich DR (2000) Microcystin-LR toxicodynamics, induced pathology and immunohistochemical localization in livers of blue–green algae exposed rainbow trout (Oncorhynchus mykiss). Toxicol Sci 54(2):365–373 Franklin NM, Rogers NJ, Apte SC, Batley GE, Gadd GE, Casey PS (2007) Comparative toxicity of nanoparticulate ZnO, bulk ZnO and ZnCl2 to a freshwater microalga (Pseudokirchneriella subcapitata): the importance of particle solubility. Environ Sci Technol 41(24):8484–8490 Galdiero S, Falanga A, Vitiello M, Cantisani M, Marra V, Galdiero M (2011) Silver nanoparticles as potential antiviral agents. Molecules 16(10):8894–8918 Gunalan S, Sivaraj R, Rajendran V (2012) Green synthesized ZnO nanoparticles against bacterial and fungal pathogens. Prog Nat Sci Mater Int 22(6):693–700
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(lemon) aqueous extract and theoretical prediction of particle size. Colloids Surf B 82(1):152–159 Raghupathi KR, Koodali RT, Manna AC (2011) Size-dependent bacterial growth inhibition and mechanism of antibacterial activity of zinc oxide nanoparticles. Langmuir 27(7):4020–4028 Rai VR, Bai AJ (2011) Nanoparticles and their potential application as antimicrobials. In: Mendez-Vilas A (ed) Science against microbial pathogens: communicating current research and technological advances, vol 1. Formatex. Res Center, Spain, pp 197–209 Reddy KM, Feris K, Bell J, Wingett DG, Hanley C, Punnoose A (2007) Selective toxicity of zinc oxide nanoparticles to prokaryotic and eukaryotic systems. Appl Phys Lett 90:2139021–2139023 Ren G, Hu D, Cheng EW, Vargas-Reus MA, Reip P, Allaker RP (2009) Characterization of copper oxide nanoparticles for antimicrobial applications. Int J Antimicrob Agents 33(6):587–590 Rice LB (2009) The clinical consequences of antimicrobial resistance. Curr Opin Microbiol 12:476–481 Romero J, Feijoo´ CG, Navarrete P (2012) Antibiotics in aquaculture—use, abuse and alternatives. In: Carvalho ED, Silva DG, da Silva RJ (eds) Health and environment in aquaculture. InTech, Croatia, p 414 Roszek B, de Jong WH, Geertsma RE (2005) Nanotechnology in medical applications: state-of-the-art in materials and devices. RIVM report 265001001/2005. Bilthoven, The Netherlands. pp 1–123 Saha S, Wang JM, Pal A (2012) Nano silver impregnation on commercial TiO2 and a comparative photocatalytic account to degrade malachite green. Sep Purif Technol 89:147–159 Seil JT, Webster TJ (2012) Antimicrobial applications of nanotechnology: methods and literature. Int J Nanomed 7:2767–2781 Singh G, Joyce EM, Beddow J, Mason TJ (2012) Evaluation of antibacterial activity of ZnO nanoparticles coated sonochemically on to textile fabrics. J Microbiol Biotechnol Food Sci 2(1):106–120 Smith P (2012) Antibiotics in aquaculture: reducing their use and maintaining their efficacy. In Austin B (ed) Infectious Disease In Aquaculture: Prevention And Control. Woodhead Publishing Limited, 80 High Street, Sawston, Cambridge, CB22 3HJ, UK. pp 1–540 Tenover FC (2006) Mechanisms of antimicrobial resistance in bacteria. Am J Infect Control 34(5):S3–S10 Vahabi K, Mansoori GA, Karimi S (2011) Biosynthesis of silver nanoparticles by fungus Trichoderma reesei (a route for largescale production of AgNPs). Insci J 1(1):65–79 Verma V (2008) Fungus disease in fish, diagnosis and treatment. Vet World 1(2):62 Wong MS, Sun DS, Chang HH (2010) Bactericidal performance of visible-light responsive titania photocatalyst with silver nanostructures. PLoS One 5(4):e10394 Wu B, Huang R, Sahu M, Feng X, Biswas P, Tang YJ (2010) Bacterial responses to Cu-doped TiO2 nanoparticles. Sci Total Environ 408(7):1755–1758
ORIGINAL PAPER
Antimicrobial activity of metal based nanoparticles against microbes associated with diseases in aquaculture P. Swain • S. K. Nayak • A. Sasmal • T. Behera S. K. Barik • S. K. Swain • S. S. Mishra • A. K. Sen • J. K. Das • P. Jayasankar
•
Received: 28 December 2013 / Accepted: 20 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The emergence of diseases and mortalities in aquaculture and development of antibiotics resistance in aquatic microbes, has renewed a great interest towards alternative methods of prevention and control of diseases. Nanoparticles have enormous potential in controlling human and animal pathogens and have scope of application in aquaculture. The present investigation was carried out to find out suitable nanoparticles having antimicrobial effect against aquatic microbes. Different commercial as well as laboratory synthesized metal and metal oxide nanoparticles were screened for their antimicrobial activities against a wide range of bacterial and fungal agents including certain freshwater cyanobacteria. Among different nanoparticles, synthesized copper oxide (CuO), zinc oxide (ZnO), silver (Ag) and silver doped titanium dioxide (Ag–TiO2) showed broad spectrum antibacterial activity. On the contrary, nanoparticles like Zn and ZnO showed antifungal activity
P. Swain (&) S. K. Nayak A. Sasmal T. Behera S. K. Barik S. K. Swain S. S. Mishra Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, India e-mail: [email protected] A. K. Sen Department of Chemical Engineering and Technology, Birla Institute of Technology-Mesra, Ranchi 835215, India J. K. Das Department of Surgery, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Technology, Bhubaneswar 751003, India P. Jayasankar Fish Genetics and Biotechnology Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, India
against fungi like Penicillium and Mucor species. Since CuO, ZnO and Ag nanoparticles showed higher antimicrobial activity, they may be explored for aquaculture use. Keywords Anti-cyanobacterial Anti-microbial Aquaculture Minimum inhibitory concentration Metal oxide Nanoparticles
Introduction The use of chemotherapeutic/antimicrobial agents is one of the most important methods of preventing and controlling infectious agents during past few decades. However, routine use of these agents leads to several adverse consequences. The rapid development of antibiotic resistance has emerged as one of the serious problems affecting all forms of life and aquatic species are no exception (Aarestrup 2005; Rice 2009). Over the years, scientific reports have been conclusive with respect to the health risk associated with indiscriminate use of antibiotics in fishes and other aquatic organisms. The indiscriminate use of antibiotics adversely affects the living systems, environment (Hektoen et al. 1995; Cabello 2006) and often leads to the development and spread of highly drug resistance pattern among pathogens. Several pathogens such as Aeromonas, Pseudomonas, Edwardsiella, Listeria, Staphylococcus, Vibrio species etc. are reported to be pathogens for both aquatic species and humans which further enhance the possibility of horizontal gene transfer (Novotny et al. 2004; Tenover 2006). Therefore, there is a need to search alternative approch to control pathogens. Diseases are the major stumbling block towards sustainable growth of the aquaculture. Fish are threatened by many pathogens, especially viruses and bacteria, often with
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serious consequences (Smith 2012; Romero et al. 2012; Leung and Bates 2013). Due to the increase in disease outbreak and the development of microbial resistance, there is an urgent need to prevent infectious agents and other harmful microorganisms without propagating antibiotic resistance pattern (Kemper 2008; Romero et al. 2012). One of the possibilities is to use nanoparticles as antimicrobial drug in aquaculture but their potential use for disease control is not fully explored yet. Recently, much attention has been paid towards the use of nanoparticles as an alternative to antibiotics due to their distinct advantages over conventional antimicrobial agents (Gunalan et al. 2012). The unique, unusual and interesting physical, chemical, and biological properties of nanometersized materials have recently attracted a great deal of interest from the scientific community. Nanoparticles with one dimension of 100 nm or less in size are now being increasingly utilised for medical applications and is of great interest as an alternative approach to control infectious agents. Among different types of nanoparticles, metals and metal oxides have elicited a great deal of interest due to their broadspectrum of antimicrobial activity, durability, high resistance, selectivity and specificity (Singh et al. 2012). Metal based nanoparticles are demonstrated to be excellent antimicrobial agent and exhibit broad spectrum antimicrobial activity against bacteria, fungi and viruses (Ren et al. 2009; Ali et al. 2011; Galdiero et al. 2011; Nasrollahi et al. 2011; Seil and Webster 2012). Therefore, the aim of the present study was to investigate the antimicrobial activity of different metal nanoparticles (both commercial and laboratory synthesized) as potential antimicrobial agent(s) for use in aquaculture practices.
Materials and methods Microorganisms Different bacterial and fungal isolates were used in the present investigation. Bacterial isolates such as Aeromonas hydrophila (FHM/AhV/2000), Edwardsiella tarda (FHM/Et/2000), Pseudomonas aeruginosa (FHM/Pa/2000), Flavobacterium branchiophilum (FHM/Fbr/2002) and other bacteria like Vibrio species (FHM/V/2000), Staphylococcus aureus (FHM/ Sa/2001), Bacillus cereus (FHM/Bc/2004) and Citrobacter species (FHM/C/2004) and four fungal isolates namely Aspergillus (FHMA/2000), Penicillium (FHMP/2000), Fusarium (FHMF/2004) and Mucor species (FHMM/2005) were used in this study. They were isolated from various disease affected freshwater fishes, characterized and maintained at Fish Health Management Division, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Odisha, India, as repository. Three cyanobacterial isolates belonging
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Table 1 Different nanoparticles used to screen antimicrobial activities in the study Sl. no.
Nanoparticles
Symbol/ formulae
Form
Size (in nm)
Commercial nanoparticles 1.
Zinc
Zn
Powder
2. 3.
Zinc oxide Zinc oxide
ZnO (C) ZnO (D)
Powder Dispersion
\50 \100 \35
4.
Copper oxide
CuO (C)
Powder
\40
5.
Iron oxide
Fe2O3
Powder
\50
6.
Iron oxide
Fe2O3
Dispersion
\30
7.
Aluminium oxide
Al2O3
Dispersion
\50
8.
Silver
Ag (D)
Dispersion
10
9.
Silver
Ag (C)
Powder
100
Synthesized (chemical and green method) nanoparticles 10.
Zinc oxide (chemical)
ZnO (S)
Powder
122.4
11.
Copper oxide (chemical)
CuO (S)
Powder
93
12.
Silver doped Titanium dioxide (chemical) Silver (green)
Ag– TiO2 Ag (S)
Powder
91.82
Suspension
58.7
13.
to Oscillatoria species (one strain) and Anabaena species (two strains), were obtained from the Department of Microbiology, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India and used in this study. Nanoparticles Both commercially available and laboratory synthesized nanaopaticles were used in the present study. Zinc (Zn); zinc oxide (ZnO); copper oxide (CuO); iron oxide (Fe2O3 powder/dispersion); aluminium oxide (Al2O3) and silver (Ag, powder/dispersion) were procured from Sigma USA. Silver (Ag), zinc oxide (ZnO); copper oxide (CuO) and silver doped titanium dioxide (Ag–TiO2) nanoparticles were synthesized in the laboratory as described below. Detailed information regarding the size and form of various nanoparticles is given in Table 1. Ag nanoparticles The green synthesis of Ag nanoparticles from AgNO3 using locally available lemon (Citrus limon) was prepared as per the procedure of Prathna et al. (2011) with slight modifications. Briefly, lemon juice was mechanically extracted from fresh lemons and then strained through a fine nylon mesh followed by centrifugation at 10,0009 rpm for 10 min at 4 °C. After centrifugation, supernatant was collected and processed for synthesis of Ag nanoparticles. The Ag nanoparticles were prepared by adding lemon extract to varying molar concentration of AgNO3 (10-2 M to 10-4 M) at different ratio of 2:3, 1:1, 3:2 and 4:1.
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Finally, solutions containing different combinations of lemon extract and AgNO3 were incubated at 60 °C in a water bath for 3 h during which change in colour of the solution from pale yellow to reddish brown took place indicating the formation of silver nanoparticles. CuO nanoparticles CuO nanoparticles were prepared as per the procedure of Lanje et al. (2010). Briefly, aqueous solution of copper acetate (0.02 M) was prepared and then 1 ml glacial acetic acid was added to the aqueous copper acetate solution. The solution was then heated to 100 °C with constant stirring. About 0.4 g of NaOH was added to above heated solution till pH reached 6–7. The formation of CuO nanoparticles was indicated by the immediate color change of the solution from blue to black. The solution was centrifuged at 15,0009 rpm for 30 min followed by three times washing with de-ionized water and finally air dried for 24 h. The dried CuO nanoparticles powder was collected in clean sterilized vials.
drop of each sample was placed on clean glass slide. After drying, the samples were sputter coated with a thin layer of 60 % gold and 40 % palladium for 30 s with 45 mA current. The morphological feature of the synthesized nanoparticles was then observed on the SEM (JEOL JSMT220A, MA) using an accelerating voltage of 5–7 kV. The average particle size of CuO, ZnO, Ag–TiO2 and Ag (only for lemon and AgNO3 ratio of 3:2) nanoparticles were determined by DLS using a laser diffraction method with a multiple scattering technique. The synthesized nanoparticles, dispersed in distilled water was first sonicated at 40 Hz for 30 min and then the samples were analyzed in a computer controlled particle size analyzer (ZETA Sizers Nano ZS, Malvern Instruments USA). Similarly, the preliminary characterization/particle size of Ag nanoparticles was determined by measuring the UV– visible spectra of the diluted synthesized solutions (1:20 in deionized water) from 200 to 600 nm range. Determination of antimicrobial activity by the disc diffusion assay
ZnO nanoparticles ZnO nanoparticles were prepared by a wet chemical method using aqueous solution of ZnCl2 (0.5 M) and NaOH (1 M) precursors as per the method of Moghaddam et al. (2009) with slight modifications. Briefly, NaOH (1 M) solution was first heated to 50 °C for 5 min and then ZnCl2 was added drop wise under high magnetic stirring. After addition of ZnCl2, the solution was sealed and stirred for further 10 min at 50 °C till white precipitate formed. ZnO nanoparticles containing pellet was washed three times with distilled water followed by twice washing with ethanol. Finally, ZnO nanoparticles were collected after drying the pellet at 50 °C. Ag doped TiO2 Ag doped TiO2 (Ag–TiO2) nanoparticles were prepared as per Saha et al. (2012) method with slight modifications. Briefly, AgNO3 was dissolved in distilled water (1.2 wt%) followed by addition of TiO2 powder (55 wt%). After addition, a paste was formed which was then dried in hot air oven at 100 °C for 2 h. This was followed by further heating in muffle furnace at 400 °C for 4 h and finally at 600 °C for 1 h. The powder was grinded in a ball mill for 2 h to decrease the particle size. Characterization of synthesized nanoparticles The morphological features of CuO, ZnO and Ag–TiO2 nanoparticles were further characterized by scanning electron microscopy (SEM). First of all, the synthesized nanoparticles were sonicated with distilled water and then one
Antimicrobial activities of nanoparticles were determined using different aquatic microbes following disc diffusion assays using the protocol of Bauer et al. (1966). Overnight grown bacterial cultures in nutrient broth (Hi-Media, Mumbai, India) (OD540 = 0.5) were individually lawn cultured on nutrient agar (Hi-Media, Mumbai, India) plates. Sterile filter paper discs containing 5 mg of nanoparticles were placed above the culture and then the plates were incubated at 30 °C for 120 h after which, the zone of inhibition were recorded. Based on the findings of inhibition zone after incubation (conducted in triplicate), the results were interpreted either as positive or negative. The nanoparticles showing inhibitory activities were further evaluated to determine minimum inhibitory concentration (MIC). Determination of minimum inhibitory concentration (MIC) The nanoparticles showing positive inhibitory activities in the preliminary disc diffusion assays were further processed to find out the MIC against different bacteria. The MIC values were calculated as per standard microbiological tube dilution method. Briefly, nutrient broth was added with individual nanoparticles ranging from 0 to 5 mg/ml (5.0, 2.5, 1.25, 1.0, 0.5, 0.62, 0.31, 0.25, 0.15, 0.1, 0.078, 0.062, 0.031, 0.025, 0.015, 0.01, 0.005, 0.007 and 0.001 mg/ml). Then 100 ll of target pathogen (at 106 CFU/ml) were added into the nanoparticles containing nutrient broth and then incubated at 30 °C overnight. After that, bacterial growth was monitored by measuring OD at 600 nm. An aliquot was streaked on nutrient agar plates. The MIC values were
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determined as the lowest concentration of nanoparticles showing complete inhibition of bacterial growth after overnight incubation. Anti-cyanobacterial assay Nanoparticles at varying concentrations (as specified for MIC value determination in antibacterial assay) were added to 10 ml of Chu-10 medium (Hi-Media, India) before inoculating with freshly grown cyanobacteria cultures. The treated cultures were incubated under diffuse florescent tube light illumination with a day/night rate of 16/8 h with gentle shaking thrice a day to avoid clumping. The MIC values were determined as the lowest concentration of nanoparticles showing complete inhibition of cyanobacteria growth after 3 weeks of culture. Effect of nanoparticles on bacterial growth Effect of laboratory synthesized CuO, ZnO and Ag nanoparticles on the growth of A. hydrophila was studied by using standard microbial methods. Briefly varying concentrations of synthesized nanoparticles (0, 0.31, 0.62, 1.25, 2.5 and 5.0 mg/ml) were separately incubated with A. hydrophila (106 to 1011 CFU/ml) overnight at 30 °C. After that the OD was recorded at 600 nm. Similar type of kinetics study was also conducted against with A. hydrophila (106 to 1011 CFU/ml) with synthesized Ag nanoparticles at various dilutions (Neat, 1/2, 1/4, 1/8, 1/16, 1/25, 1/50, 1/100) of lemon extract and AgNO3 as controls. Anti-fungal assay The antifungal activity of different nanoparticles was determined as per the disc diffusion method. Briefly, a loop full of fungal mycelia was inoculated at the center of a Sabouraud dextrose agar (SDA, Hi-Media, India) plates and then sterile filter paper discs containing individual nanoparticles suspension at varying concentrations were placed in the periphery. The concentrations used were same as specified for MIC value determination in antibacterial assay. The plates were incubated at room temperature for 7 days and were monitored for inhibitory zone. The MIC values were determined as the lowest concentration of nanoparticles showing complete inhibition of fungal growth after 7 days of incubation.
Fig. 1 The SEM micrographs showing the surface morphology of synthesized a CuO, b ZnO and c Ag–TiO2
Results Characterization of synthesized nanoparticles The surface morphology of the laboratory synthesized CuO, ZnO and Ag–TiO2 nanoparticles are shown in the
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Fig. 1a–c. SEM micrographs of the CuO showed free and spherical forms of nanoparticles. On the contrary, ZnO nanoparticles were found to be aggregated, forming a microstructure, while Ag–TiO2 nanoparticles were found to be densely packed spherical forms.
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Fig. 3 The UV spectra of green synthesized Ag nanoparticles using different ratio of AgNO3 and Lemon
mean percentage of particles were maximum (32.9 %) with peak at 93 nm for synthesized CuO while 83.8 % of ZnO were 122.4 nm in size. Ag–TiO2 also revealed a broad size distribution with particles in the size range of 43.8 to 190.1 nm with a mean percentage of particles maximum (15 %) at a peak value of 91.82 nm. The formation of silver nanoparticles obtained from varying the mixing ratios between AgNO3 and lemon extract were also confirmed by UV–visible spectroscopy (Fig. 3). Among four different combinations of lemon extract and AgNO3 used to prepare Ag nanoparticles, the peak was obtained at 460 nm in 4:1 ratio, 420 nm in 3:2 ratio, 439 nm in 1:1 ratio and 441 nm in 2:3 ratio of lemon extract and AgNO3. The particles size of the Ag nanoparticles (of 3:2 ratio) as determined from DLS study ranged from 43 to 140 nm with peak at 58.7 nm. Anti-bacterial activity
Fig. 2 The average particle size of various laboratory synthesized nanoparticles as determined by DLS. a CuO, b ZnO, c Ag–TiO2 and d Ag (Lemon 3: 2 AgNO3)
The size distribution pattern of the laboratory synthesized (CuO, ZnO, Ag–TiO2 and Ag nanoparticles) as determined by DLS study is shown in Fig. 2a–d. The particle distribution curve was broad for CuO whereas that of ZnO were more uniform with a narrow range of distribution. The particle size of CuO and ZnO ranged from 58.77 to 141.8 and 105.7 to 122.4 nm, respectively. The
Different nanoparticles exhibited antibacterial activities against various bacterial isolates by disc diffusion assay (Fig. 4a–d). Among different nanoparticles, commercial nanoparticles like ZnO and CuO were found to inhibit the bacteria but Zn, Ag, Fe2O3, and Al2O3 nanoparticles failed to inhibit any of the tested isolates. But in case of commercial ZnO nanoparticle treatment, over growth of B. cereus, P. aeruginosa and Vibrio species, was recorded upon prolonged incubation upto 120 h. Likewise, the chemical synthesized nanoparticles, ZnO and CuO were found to inhibit all bacterial isolates irrespective of incubation period. However, chemical synthesized Ag–TiO2 nanoparticles only inhibited A. hydrophila, E. tarda, F. branchiophilum, S. aureus and Citrobacter species but on
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Fig. 4 Disc diffusion assays showing zone of inhibition exhibited by various nanoparticles against different bacterial isolates. a B. cereus; b P. aeruginosa; c E. tarda; d A. hydrophila. Numerical numbers in
the individual disc of individual plate (a–d) represent different types of nanoparticles 1 CuO(S); 2 CuO(C); 3 ZnO(S); 4 ZnO(D); 5 Ag; 6 Zn 7 Al2O3; 8 Fe2O3
prolonged incubation upto 120 h, overgrowth was recorded (Table 2). On the other hand, Ag nanoparticles prepared by different combinations of lemon and AgNO3 exhibited antibacterial activities. With lemon extract as a control treatment, highest zone of inhibition against E. tarda and S. aureus was recorded in lemon extract diluted with water at 4:1 ratio followed by 3:2, 1:1 and 2:3 ratios. However, the zone of inhibition was highest against P. aeruginosa at 2:3 dilution while no variation in the zone size could be recorded at any dilutions of lemon extract against A. hydrophila. Similarly, in AgNO3 control group, AgNO3 at 10-3 M and 10-4 M did not exhibit any antibacterial activity since no zone of inhibition against any of the tested bacteria could be observed. However, 10-2 M AgNO3 exhibited highest inhibitory zone against all bacteria. On the contrary, highest zone of inhibition was recorded in the
Ag nanoparticles prepared from lemon 10-2 M and AgNO3 at 3:2 ratio (Table 3).
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MIC values determination The MIC values of various commercial and laboratory synthesized nanoparticles against different bacterial pathogens is given in Table 4. The MIC values of commercial CuO and laboratory synthetized ZnO nanoparticles against most of the pathogens were found to be more than 5 mg/ml. On the contrary, chemically synthesized nanoparticles, CuO nanoparticles showed lower MIC values against most of the pathogens as compared to that of ZnO and Ag–TiO2 nanoparticles. The MIC values were found to be more than 5 mg/ ml for ZnO and Ag–TiO2 nanoparticles . The MIC values of laboratory synthesized Ag nanoparticles against different bacterial isolates are given in
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Table 5. The least MIC values against various bacteria were recorded when Ag nanoparticles were prepared at 3:2 dilution of Lemon: AgNO3 while highest MIC values were recorded when Ag nanoparticles were prepared at 2:3 dilution of Lemon extract: AgNO3. Anti-cyanobacterial activity Nanoparticles were found to inhibit the growth of Oscillatoria and Anabaena species (Table 6) but the minimum inhibitory dose with regard to nanoparticles varied. Among the nanoparticles, Fe2O3 was found to be most effective nanoparticles with least MIC values of 0.015 and
Table 2 Zone of inhibition exhibited by various nanoparticles against different bacterial isolates Sr. no
Nanoparticles
Pathogens 1
2
3
4
5
6
7
8
Commercial nanoparticles 1.
Zinc (Zn)
-
-
-
-
-
-
-
-
2.
Zinc oxide (ZnO) (C)
?
?
?
?
?
-
-
-
3. 4.
Zinc oxide (ZnO) (D) Copper oxide (CuO) (C)
? ?
? ?
? ?
? ?
? ?
? ?
? ?
? ?
5.
Iron oxide (Fe2O3)
-
-
-
-
-
-
-
-
6.
Iron oxide (Fe2O3) (D)
-
-
-
-
-
-
-
-
7.
Aluminium oxide (Al2O3)
-
-
-
-
-
-
-
-
8.
Silver (Ag) (D)
-
-
-
-
-
-
-
-
9.
Silver (Ag) (C)
-
-
-
-
-
-
-
-
Synthesized nanoparticles
0.062 mg/ml against two Anabaena species while that of Oscillatoria species was 0.031 mg/ml. Apart from Fe2O3, CuO(S), Ag(S) and ZnO(D) were also effective with low MIC values against all the three cyanobacteria. However, among the three isolates of cyanobacteria, Oscillatoria showed higher resistance towards nanoparticles and the MIC values were found to be higher than 5 mg/ml against ZnO(C), Ag(C) and Al2O3. Both the Anabaena species were completely inhibited by CuO(C), Ag–TiO2, and Al2O3 with MIC values of 0.062, 0.078 and 0.5 mg/ml respectively. Effect of nanoparticles on bacterial growth Effect of different concentration of laboratory synthesized CuO nanoparticles on A. hydrophila is shown in Fig. 5a. CuO nanoparticles were found to reduce the growth of A. hydrophila in a concentration dependent manner. Complete inhibition of A. hydrophila upto 1010 CFU/ml was recorded for varying concentration of CuO nanoparticles ranging from 0.31 to 5 mg/ml. However, growth of A. hydrophila at 1011 CFU/ml was recorded even at treatment of CuO nanoparticles at 5 mg/ml. But, less growth was recorded as Table 4 MIC values of various commercial and laboratory synthesized nanoparticles against different bacterial agents Sr. no
Bacteria
MIC values (mg/ml)
1
Aeromonas hydrophila
ZnO (D)
ZnO CuO (S) (C)
CuO (S)
Ag– TiO2
0.25
2.5
1.2
0.31
1.2
10.
Zinc oxide (ZnO) (S)
?
?
?
?
?
?
?
?
2
Edwardsiella tarda
0.62
0.31
0.62
2.5
0.25
11.
Copper oxide (CuO) (S)
?
?
?
?
?
?
?
?
3
Bacillus cereus
[5
[5
[5
1.2
[5
12.
Silver–Titanium dioxide (Ag–TiO2)
?
?
?
?
?
-
-
-
4
Citrobacter species
1.2
0.62
2.5
1.2
0.25
5
1.2
[5
[5
2.5
0.25
Ag (S)
?
Flavobacterium branchiophilum
0.31
13.
?
?
?
?
?
?
?
1: A. hydrophila 2: Citrobacter species 3: E. tarda 4: F. branchiophilum 5: S. aureus 6: Vibrio species 7: B. cereus 8: P. aeruginosa
6
Staphylococcus aureus
[5
1.2
[5
0.16
7
Vibrio species
[5
[5
[5
0.31 [5
? Inhibition, - No inhibition
8
Pseudomonas aeruginosa
[5
[5
[5
0.16 [5
Table 3 Zone of inhibition (in mm) against different bacterial agents exhibited by green synthesized Ag nanoparticles
Lemon: AgNO3 ratio
A. hydrophila
E. tarda
S. aureus
P. aeruginosa
4:1
3:2
1:1
2:3
4:1
3:2
1:1
2:3
4:1
3:2
1:1
2:3
4:1
3:2
1:1
2:3
10-2
18
21
18
20
10
16
11
16
20
25
21
24
16
16
17
18
10-3
9
9
9
9
10
10
10
10
10
10
10
9
9
8
8
9
10-4
9
9
9
9
11
10
9
9
10
10
9
9
8
8
8
9
AgNO3 (10-2)
12
20
18
18
10
14
13
17
18
23
21
24
12
14
16
17
AgNO3 (10-3)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
AgNO3 (10-4)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Lemon extract
8
8
8
8
11
10
9
9
10
10
9
9
8
7
8
9
Control groups
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the fungi were found to be 2.5 mg/ml. Similarly, both forms of ZnO nanoparticles i.e., ZnO(S) and ZnO(D) were also found to inhibit Penicillium and Mucor species with MIC value of 2.5 and 5.0 mg/ml, respectively. On the contrary, Zn and ZnO nanoparticles, the MIC values were higher than 5.0 mg/ml for Aspergillus and Fusarium species. Apart from these nanoparticles, others nanoparticles like CuO(S), CuO(C), Fe2O3, Al2O3 Ag(C) and Ag(S) did not exhibit antifungal activity up to 5 mg/ml against any of the tested fungi (Table 7).
compared to control group. Similar types of inhibitory pattern against A. hydrophila was also recorded for ZnO nanoparticles (Fig. 5b). On the other hand, Ag nanoparticles synthesized from AgNO3 using lemon extract irrespective of dilution completely inhibited the growth of A. hydrophila up to 1010 CFU/ml while growth was recorded in the group treated with A. hydrophila (1011 CFU/ml) Ag nanoparticles at 1/100 and 1/50 dilution. On the contrary, complete inhibition of A. hydrophila growth was recorded at 1/2 dilution of lemon extract while that of AgNO3 (10-2 M) was observed at 1/8 dilution. At 1/50 dilution, lemon and AgNO3 (10-2 M) showed very low antibacterial activity with the growth pattern similar to that of control (Fig. 5c).
Discussion The field of nanotechnology is rapidly expanding and used in various areas, such as health management, food and feed, environmental aspects and agricultural practices (Roszek et al. 2005; Bouwmeester et al. 2007; Kiruba Daniel et al. 2013). Over the years, nanoparticles have achieved unprecedented success in all areas of their applications for the benefit of society. With unique
Anti-fungal activity Among all the tested nanoparticles, Zn and ZnO showed antifugal activity against Penicillium and Mucor species (Fig. 6a, b). Zn nanoparticles, were found to inhibit Penicillium and Mucor species and the MIC values against both Table 5 MIC values (in mg/ml) of green synthesized Ag nanoparticles against different bacterial agents
Table 6 MIC values (in mg/ ml) of various commercial and laboratory synthesized nanoparticles against different cyanobacteria
Table 7 MIC values (in mg/ ml) of various commercial and laboratory synthesized nanoparticles against different fungi
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Sr. no
Bacteria
Minimum inhibitory concentration (mg/ml) Lemon:AgNO3 (1:1)
Lemon:AgNO3 (2:3)
Lemon:AgNO3 (3:2)
1
Aeromonas hydrophila
2.7
6.4
0.21
2
Edwardsiella tarda
2.7
6.4
0.21
3
Bacillus cereus
2.7
6.4
0.21
4
Citrobacter species
2.7
6.4
0.21
5
Flavobacterium branchiophilum
2.7
6.4
0.21
6
Staphylococcus aureus
2.7
1.6
0.21
7
Vibrio species
1.3
1.6
0.1
8
Pseudomonas aeruginosa
1.3
1.6
0.1
Sr. no
Species
Minimum inhibitory concentration (mg/ml) Fe2O3
Zn
ZnO (D)
ZnO (S)
CuO (C)
CuO (S)
Ag (C)
Ag (S)
Al2O3
Ag–TiO2
1
Anabaena
0.015
0.12
0.078
0.25
0.062
0.062
[5
0.062
0.5
0.078
2
Anabaena
0.062
0.25
0.078
0.12
0.062
0.031
[5
0.062
0.5
0.078
3
Oscillatoria
0.031
0.25
0.015
[5
0.12
0.078
[5
0.078
[5
0.25
Sr. no
Species
Minimum inhibitory concentration (mg/ml) Fe2O3
Zn
ZnO (D)
ZnO (S)
CuO (C)
CuO (S)
Ag (C)
Ag (S)
Al2O3
Ag–TiO2
2.5
2.5
2.5
[5
[5
[5
[5
[5
[5
1
Penicillium
[5
2
Mucor
[5
2.5
5
5
[5
[5
[5
[5
[5
[5
3
Fusarium
[5
[5
[5
[5
[5
[5
[5
[5
[5
[5
4
Aspergillus
[5
[5
[5
[5
[5
[5
[5
[5
[5
[5
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Fig. 5 Effects of synthesized nanoparticles on the growth of A. hydrophila at 106 to 1011 CFU/ml. (a CuO; b ZnO; c: Ag)
properties like high surface volume ratio, ability to withstand intensive processing conditions, depressed melting temperature, conductivity, magnetism and light absorption, nanoparticles have gained tremendous advantages as compared to their large sized counterparts (Feynman 1991). Recently, metals and/or their oxide nanoparticles by virtue of their physico-chemical properties have emerged as promising antimicrobial agents. These nanoparticles can adopt various mechanisms like inhibition of the synthesis of functional biomolecules or impeding normal cellular activities to kill pathogens (Rai and Bai 2011).
Fishes are susceptible to a wide variety of microbial agents among which bacterial diseases are most prevalent causing severe economic losses throughout world. Numerous species of bacteria are capable of causing diseases in fish and the emergence of antimicrobial resistance by pathogenic bacteria is also a major concern. Similarly, fungal infections are common, especially under stress conditions and poor water quality (Verma 2008). Furthermore, many times cyanobacterial blooms are affecting aquaculture throughout world and cyanotoxin like microcystins is highly toxic to fish at micrograms level (Carbis et al. 1996; Fischer et al. 2000; Malbrouck and Kestemont 2006; Ernst 2008). Therefore, as an alternate approach, we have evaluated a battery of nanoparticles both commercial and laboratory synthesized, for their antimicrobial activity against a broad range of microbial agents. We have synthesized the Ag–TiO2, CuO, ZnO and Ag nanoparticles in laboratory. The mean percentage of particles were maximum (32.9 %) with peak at 93 nm for synthesized CuO, 83.8 % of ZnO nanoparticles were of 113 nm in size and Ag–TiO2 revealed a maximum (15 %) at a peak value of 91.82 nm. Similarly, we also synthesized Ag nanoparticles using lemon extract as a suitable reducing agent. Ag nanoparticles synthesized in 4 combinations of lemon extract and AgNO3 showed peak at range from 420 to 460 nm and was in accordance to earlier report (Prathna et al. 2011). The UV-visible spectra of indicated size of Ag nanoparticles using lemon extract and AgNO3 (10-2 M) in the ratio 3:2 to vary in 50.78 to 78.82 nm which was also corroborated from the DLS data. Among various nanoparticles, ZnO, CuO, Ag and Ag– TiO2 demonstrated antimicrobial activities. On the contrary, commercial Ag, Fe2O3 and Al2O3 showed no antibacterial activity upto a concentration of 5 mg/ml. In this study, we have also demonstrated antibacterial activity of CuO nanoparticles against all the tested bacterial agents. Similar types of antibacterial activity of CuO nanoparticles have been demonstrated against different bacterial species by earlier researchers (Baek and An 2011; Azam et al. 2012; Abboud et al. 2013). However, among the CuO nanoparticles, the particle size of commercial CuO was \40 nm as compared to higher size (B93 nm) of the laboratory synthesized CuO but synthesized CuO nanoparticles exhibited better antimicrobial activities with least MIC values than commercial CuO nanoparticles. The present findings differ from the earlier findings of Azam et al. (2012) who had reported the size dependent antimicrobial activity of CuO nanoparticles. They have also recorded CuO nanoparticles of 20 (±1.24) nm size to inhibit significantly various pathogens as compared to the higher sized CuO nanoparticles. Such difference in the antibacterial activity might be attributed to the difference in dissolving properties and subsequent aggregation of
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Fig. 6 Antifungal activity of Zn and ZnO nanoparticles at different concentration against a Penicillium and b Mucor species after 7 days of inoculation. Numerical numbers in the individual disc of individual
plate (6a, b) indicate the concentration of nanoparticles: 1 Zn (5.0 mg/ml); 2 Zn (2.5 mg/ml); 3 ZnO(S) (5.0 mg/ml); 4 ZnO (S) (2.5 mg/ml); 5 ZnO(D) (2.5 mg/ml); 6 ZnO (D) (5.0 mg/ml)
commercial CuO nanoparticles in the aqueous environment could have created hindrance in direct interaction between particles and microorganisms (Wu et al. 2010). Similarly, ZnO nanoparticles have a wide range of applications in various fields due to their unique and superior physical and chemical properties compared with bulk ZnO. Earlier studies showed size dependent inhibitory activity of nanoparticles including ZnO (Jones et al. 2008; Nair et al. 2009; Raghupathi et al. 2011). ZnO nanoparticles of 13 nm size were found to inhibit S. aureus growth at 0.081 mg/ml (Reddy et al. 2007) while ZnO nanoparticles of size 60 nm at 0.4 mg/ml only reduced 50 % of S. aureus (Jones et al. 2008). Herein, we have also found higher MIC value of 1.2 mg/ml against S. aureus by ZnO nanoparticles of 122.4 nm size. This could be attributed to better solubility of synthesized ZnO nanoparticles in aqueous medium. However, we failed to record inhibitory activity of commercial ZnO of \100 nm against bacteria like Vibrio species, B. cereus and P. aeruginosa where the MIC values were found to be more than 5 mg/ml. Such difference could be attributed to the species specific differences. The antimicrobial effect of Ag nanoparticles has already been demonstrated in several studies (Kvitek et al. 2008; Dror-Ehre et al. 2009; Panacek et al. 2009; Kim et al. 2011). We have synthesized Ag nanoparticles using lemon extract as a suitable reducing agent and demonstrated excellent antibacterial activities against a broad range of pathogens with very low MIC values. However, commercial Ag did not show antibacterial activity and the MIC values were more than 5 mg/ml against all bacterial isolates which might be due to their agglomerating nature during commercial preparations as compared to green synthesized method. However, the Ag synthesized using
lemon failed to exhibit any anti-fungal activities. Ag–TiO2 nanoparticles exhibited antibacterial activity against specific bacteria. The antimicrobial activity of Ag–TiO2 may be attributed to the facts that the small particle size of Ag? could enhance its solubility and the presence of extracellular Ag? could interfere with the intracellular Ca? metabolism and cause cellular damage (Korbekandi and Iravani 2012). Further TiO2 might be also responsible in enhancing the activity via photocatalysis or generation of reactive oxygen species (Wong et al. 2010). In this study, we have also included four fungal species that are associated with Aquaculture. Penicillium and Mucor species were sensitive to Zn and ZnO. Limited information’s are available on the antifungal activity of ZnO nanoparticles (Chitra and Annadurai 2013). However, available reports indicate the antifungal activities of ZnO nanoparticles against different fungi such as Penicillium, Aspergillus, Fusarium etc. from different sources (He et al. 2011; Chitra and Annadurai 2013; Dimkpa et al. 2013). He et al. (2011) also observed inhibitory activity of ZnO of 70 (±15) nm at minimum of 3 mmol/l (&0.244 mg/ml) against Penicillium expansum. Gunalan et al. (2012) demonstrated that higher sized ZnO (40 nm) to execute enhanced antimicrobial activity against bacteria and fungus when compared to ZnO of lower particle size (25 nm). Herein, ZnO nanoparticles were found to be effective against Penicillium and Mucor species. But the findings are contradictory to that of Chitra and Annadurai (2013) who demonstrated the antifungal activity of ZnO nanoparticles (100–146 nm) against Aspergillus niger with a maximum inhibition concentration of 0.4 mg. So there may be species specific inhibitory activities exerted by ZnO. Also effectiveness of nanoparticles increases with increasing particle
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dose, treatment time and synthesis method. Additionally variation in the particle size and surface area to volume ratio of the synthesized ZnO nanoparticles are possibly contributed to better antimicrobial activity. ZnO was found to inhibit the growth of Penicillium and Mucor species at 2.5 and 5 mg/ml, respectively. The inhibitory activity of ZnO nanoparticles against different fungi is in agreement with earlier reports (Lipovsky et al. 2011). However, Aspergillus and Fusarium species were resistant upto 5 mg/ml. The antifungal activity of ZnO may be due to suppression of extracellular enzymes and metabolites that helps its survival when exposed to stress as demonstrated by fungi like Aspergillus fumigatus, Trichoderma reesei (Bhainsa and D’ Souza 2006; Vahabi et al. 2011). While a bulk of informations are already available on the effect of nanoparticles on bacteria and fungi, till date only a few reports are available on the effect of nanoparticles on unicellular green algae (Franklin et al. 2007; Dash et al. 2012). Among different cyanobacterial species, Anabaena and Oscillatoria species are the two common microcystins producing toxic cyanobacteria found mostly in freshwater system. Here, we have demonstrated that nanoparticles like Fe2O3, CuO(S), Ag(S) and ZnO(D) to be very effective against both the species of Anabaena and Oscillatoria species. It is therefore, summarized from the present study that nanoparticles, especially synthesized CuO, ZnO and Ag have excellent antimicrobial activity and could be further explored as substitute of antibiotics in aquaculture use. However, there is also a need to understand the detailed antimicrobial mechanism(s) of various metal nanoparticles against other microbes dwell in the aquaculture system and an in-depth study is further required for accessing the risks associated with them before their application in aquaculture. Acknowledgments The authors are thankful to Indian Council of Agricultural Research (ICAR), New Delhi for funding the project under the National Fellow Scheme. The authors are also thankful to the Director of Central Institute of Freshwater Aquaculture, Kausalyaganga, Odisha, India for providing necessary facility to carry out the present work. Conflict of interest
None.
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