www.ietdl.org Published in IET Nanobiotechnology Received on 9th April 2012 Revised on 13th September 2012 Accepted on 19th September 2012 doi: 10.1049/iet-nbt.2012.0008
ISSN 1751-8741
Size-dependent antimicrobial response of zinc oxide nanoparticles Loganathan Palanikumar1, Sinna Nadar Ramasamy1, Chandrasekaran Balachandran2 1
Crystal Growth Centre, Anna University, Chennai 600025, India Division of Microbiology, Entomology Research Institute, Loyola College, Chennai 600034, India E-mail: [email protected]
2
Abstract: Antibacterial and antifungal activities of zinc oxide nanoparticles (ZnO NPs) were investigated against infectious microorganisms. ZnO NPs were prepared by wet chemical precipitation method varying the pH values. Particle size and morphology of the as-prepared ZnO powders were characterised by X-ray diffraction, Fourier transform infrared spectroscopy and transmission electron microscope. The zone of inhibition by NPs ranged from 0 to 17 mm. The lowest minimum inhibitory concentration value of NPs is 25 µg.ml−1 against Staphylococcus epidermidis. These studies demonstrate that ZnO NPs have wide range of antimicrobial activities towards various microorganisms. The results obtained in the authors’ study indicate that the inhibitory efficacy of ZnO NPs is significantly dependent on its chosen concentration and size. Significant inhibition in antibacterial response was observed for S. epidermidis when compared with control antibiotic.
1
Introduction
As particles are reduced from a micrometre to nanometer size, the resulting properties can change dramatically. For example, electrical conductivity, hardness, active surface area, chemical reactivity and biological activity are all known to be altered. Medicinal sciences are investigating the use of nanotechnology to improve medical diagnosis and treatments [1–3]. The bactericidal effectiveness of metal NPs has been suggested to be because of both particle size and high surface-to-volume ratio. Such characteristics should allow them to interact closely with bacterial membranes, rather than the effect being solely because of the release of metal ions [4]. Inorganic antibacterial agents are more stable at high temperatures and pressures compared with the organic materials, and the metallic oxide powders could be suggested as powerful antimicrobial agents [5]. Zinc oxide is traditionally used for the photocatalytic oxidation of organic and inorganic pollutants and sensitisers for the photodestruction of cancer cells, bacteria and viruses via oxidative damage [6–8]. NPs generally produces toxic effects with plasma proteins, transient inflammatory, cell injury effects leading to inflammation and fibrosis. The NPs have shown elevated blood biochemical parameters, accumulation of foamy alveolar macrophages, degenerated alveolar macrophages indicating alveolar lipoproteinosis. In humans, some types of NPs which are used for cosmetics etc. may result in skin disease. However, ZnO#ZnS quantum dots heterojunctions have been reported to enhance transportation of flavonoid glycosides in blood [7, 8]. Hence, it is appropriate to assume that ZnO is non-toxic and so it has been taken for this research study. IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
ZnO NPs are widely used in many consumer products like cosmetics, toothpaste, textiles and skin lotions [9]. Staphylococcus aureus and S. epidermidis are natural inhabitants of human and animal skin, but it can sometimes cause infections that affect many organs [10]. The pathogenic bacteria Klebsiella pneumoniae and Enterobacter aerogenes are the major causative agents of nosocomial infections [11]. Since 1980s, methicillin-resistant S. aureus (MRSA) has been commonly linked with hospital-associated infections [12]. Paratyphoid fever is caused by Salmonella enterica serotypes Paratyphi A, Paratyphi B or Paratyphi C. As per literature, an estimated 5 400 000 cases of paratyphoid fever occurred globally in 2000 [13]. Serotype Paratyphi B var. L (+) tartrate (+) causes a typical Salmonella gastroenteritis instead of enteric fever [14]. Candida albicans and Malassezia pachydermatis are the most frequent human and animal pathogens [15, 16]. The antibacterial and antifungal activities of bulk ZnO powders and ZnO NPs have been demonstrated already [12, 17]. However, little is known about the activity of ZnO NPs in the range 15–50 nm range towards the infectious microorgansims. Towards this purpose, evaluation has been made on the antibacterial and antifungal effect as a function of synthesised ZnO NPs size based on the inhibition zone in the disk diffusion tests with determining minimum inhibitory concentration (MIC).
2 2.1
Materials and methods Synthesis of ZnO NPs
ZnO NPs were synthesised with a slight modification suggested by Wu et al. [18] from aqueous solutions of zinc 111
& The Institution of Engineering and Technology 2014
www.ietdl.org nitrate [Zn (NO3)2.6H2O; purchased from Fischer Chemicals, Mumbai, India; purity 96%] and hexamethyltetramine (HMT; C6H12N4; purchased from Qualigens chemicals Ltd., Mumbai, India; purity 99%). The two chemicals were mixed separately with milli-Q water to a concentration of 0.05 M for the [Zn (NO3)2] solution, and 1.5 M for the HMT solution. The separate solutions are stirred for 30 min each, and then mixed with 130 rpm stirring. The solutions was adjusted to the desired pH (5.0, 6.0 and 7.2) and heated to 80°C for 45 min. The product is collected by centrifugation (Compufuge, Remi Electrotechnik Limited, Thane, India). The ammonium hydroxide solution (1 N) was added to pH 5.0 synthesised solutions to enhance the formation of ZnO at 80°C [19]. 2.2
Particle characterisation
Powder X-ray diffraction (XRD, Seifert, JSO-DE BYEFLEX 2002, Germany) was utilised to identify the crystalline phase composition and purity. The phase was found to be hexagonal and no impurity peaks were found. The morphology and grain size of the ZnO were observed by TECNAI G2 Model T-30 S-twin high-resolution transmission electron microscopy (HRTEM). The quality of the ZnO NPs was analysed by Fourier transform infrared spectroscopy (FTIR, Perkin Elmer Spectrum One). 2.3
Microbial organisms
The following bacteria and fungi were used for the experiment. Bacteria: Salmonella paratyphi B, K. pneumoniae MTCC 109, Bacillus subtilis MTCC 441, E. aerogenes MTCC 111, Staphylococcus epidermidis MTCC 3615, Methicillin resistant-MRSA. The reference cultures were obtained from Institute of Microbial Technology (IMTECH; Chandigarh, India 160 036); fungi: C. albicans MTCC 227 and M. pachydermatis were obtained from the Department of Microbiology (Christian Medical College, Vellore, Tamil Nadu, India). 2.4
Antimicrobial assay
Antibacterial and antifungal activities were carried out using disc-diffusion method [20, 21]. Petri plates were prepared with 20 ml of sterile Mueller Hinton agar (MHA) (Himedia, Mumbai). The test cultures were swabbed on the top of the solidified media and allowed to dry at room temperature for 10 min. The suspension of NPs (in milli-Q water) was sonicated to prepare required suspensions such as 50, 100 and 200 µg.ml−1 added to each well (diameter of 8 mm) separately. Preliminarily, a broad range of concentrations (10–200 µg.ml−1) were chosen for antimicrobial assay (data not shown). Based on those results, three concentrations were chosen for the present work as a function of size. The suspension of NPs were loaded on the wells of the medium and left for 30 min at room temperature for diffusion. Negative control was prepared using respective solvents. Streptomycin (25 µg/ disc; purchased from Himedia Chemicals Ltd., Mumbai, India) was used as positive control against bacteria. Ketoconazole (25 µg/disc; purchased from Himedia Chemicals Ltd., Mumbai, India) was used as positive control for fungi. The plates were incubated for 24 h at 37°C for bacteria and for 48 h at 28°C for fungi. Zones of inhibition were recorded in millimetres by repeating the experiment thrice for each concentration. 112 & The Institution of Engineering and Technology 2014
2.5
Minimum inhibitory concentration
MIC studies of the NPs were performed according to the standard reference methods for bacteria [22], for filamentous fungi [21] and yeasts [23]. The required concentrations (3.125, 6.25, 12.5, 25, 50, 100 and 200 µg. ml−1) of the NPs were dispersed in milli-Q water and diluted to give serial two-fold dilutions that were added to each medium in 96 well plates. A volume of 100 µl (inoculum) from each well was inoculated. The antifungal agents ketoconazole for fungi and streptomycin for bacteria were included in the assays as positive controls. For fungi, the plates were incubated for 48–72 h at 28°C and for bacteria the plates were incubated for 24 h at 37°C. The MIC for fungi was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. 5 µl of tested broth was placed on the sterile MHA plates for bacteria and incubated at respective temperatures. The MIC for bacteria was determined as the lowest concentration of the compound inhibiting the visual growth of the test cultures on the agar plate. 2.6
Statistical analysis
The differences in antimicrobial activity of ZnO NPs in comparison with control were assessed by one-way analysis of variance [24]. Dunnett’s post hoc test was employed to compare the significant difference between control and different exposure concentrations. This statistical analysis was carried out using computer-assisted software program Graph Pad Prism version 5.0. Other statistical analysis was carried out using Microsoft Office Excel 2003.
3
Results
The present study employed a low-temperature synthesis method to prepare ZnO NPs. Zinc nitrate and HMT solutions were mixed at 80°C for 45 min at different pH and a precipitation reaction occurred, after the pH was 8.2. The XRD pattern of synthesised ZnO NPs demonstrated that the ZnO is crystalline in nature, and the diffraction peaks matched very well with a hexagonal zincite (wurtzite) phase of ZnO (Fig. 1). The diffraction pattern and inter-planar spacing closely matched those in the standard diffraction pattern of ZnO (Powder diffraction file ICDD
Fig. 1 XRD pattern of ZnO NPs prepared at different pH IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
www.ietdl.org 36-1451 a = 3.249 Å and c = 5.206 Å). The XRD peaks show (100), (002), (101), (102), (110), (103), (200) and (201) reflection lines of hexagonal zincite phase of ZnO particles. No characteristic peaks of any impurities were detected suggesting good quality and widened peak were detected. The particle size based on broadening was analysed by Scherrer formula, modified form of Williamson–Hall analysis and size–strain plot method. The crystalline size can be calculated using the following equation 2 2 dhkl bhkl cosu = K/D dhkl bhkl cosu + (1/2)2 where K is the constant that depends on the shape of the particles. The particle size determined from the slope of linearly fitted data and the root of the y-intercept gives the strain [25].
The quality of the as-synthesised product without any heat treatment was analysed by the FTIR spectroscopy. FTIR confirm the formation of ZnO. Fig. 2 shows the FTIR spectrum acquired in the range of 400–4000 cm−1. The band at 535 cm−1 corresponds to the stretching vibration of Zn–O bond. The broadening corresponding to Zn–O peak at 535 cm−1 is broadened. This may be because of the nanocrystalline nature of the compound. The broad absorption bands in the range 3900–2350 and 1637 cm−1 correspond to the presence of the surface hydroxyl groups [26]. C–OH stretching (1387 cm−1) are detected from the FTIR spectrum [27]. The band at 1387 cm−1 corresponds to the CH2 deformation and absorption bands at 1235–1125 cm−1 are responsible for CN stretch. The band at 998 and 897 cm−1 corresponds to CH2 rock [28]. Although these extra peaks because of the starting material hexamethylenetetraamine appear in the as-prepared material, the tetramethyl amine is expected to go-off on heat treatment, before we go for antimicrobial and MIC studies. The morphology and size distribution of the ZnO NPs prepared at various pH are shown in Fig. 3. It can be clearly observed that the size of NPs was reduced with
Fig. 3 FTIR spectrum of as-prepared ZnO NPs (38 nm)
decreasing pH. The number of hexagonal-shaped NPs increased as the pH decreased. The TEM images confirmed the formation of hexagonal structure of ZnO and are in agreement with the XRD results. The particle size of the ZnO nanograins prepared at different pH was 38 ± 2; 25 ± 4 and 15 ± 4 nm in as-prepared (pH 7.2), pH 6.0 and pH 5.0, respectively. Modified Scherrer formula gives 32 ± 4; 25 ± 4 and 15 ± 4 nm, respectively, for the above samples. 3.1
The antibacterial and antifungal activity of ZnO NPs was compared for infectious Gram-positive and Gram-negative bacteria and fungus. The results of antimicrobial activity of different size of ZnO NPs were shown in Table 1 and Figs. 4a–h. The zone of inhibition (in mm) reflects the magnitude of susceptibility of the microorganism. The strains susceptible to NPs exhibit larger zone of inhibition, whereas resistant strains exhibit smaller or no zone of inhibition. Significant difference between control antibiotics (streptomycin and ketoconazole) were observed for all the NPs treatment, whereas insignificant zone of inhibition were observed for S. epidermis. No antimicrobial response was observed for 25 and 38 nm ZnO NPs against K. pneumonia. Table 2 presents the MICs of ZnO NPs for infectious microorganisms. Highest MIC response was observed for S. epidermis.
4
Fig. 2 TEM images of ZnO NPs prepared at different pH a As prepared b pH6.0 c pH5.0 IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
Antimicrobial and MIC studies
Discussion
The re-emergence of infectious diseases poses a serious threat to public health worldwide, and the increasing rate of the appearance of antibiotic-resistant strains in a short period of time within both Gram-positive and Gram-negative bacteria and fungal microorganisms is a major public health concern [12]. Alternative therapeutics to control and prevent the spread of infections in both community and hospital environments are required [29]. ZnO NPs are much more effective agents in controlling the growth of various microorganisms [12, 30, 31]. Our preliminary studies show that the reduced particle size had greater efficacy in inhibiting the growth of microorganisms. Similarly, metals and metal oxides such as ZnO NPs are known to be toxic to host human and 113
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www.ietdl.org Table 1 Antimicrobial activity of ZnO NPs (zone of inhibition in mm; 200 µg.ml−1) Organism
15 ± 4 nm ZnO NPs
25 ± 4 nm ZnO NPs
38 ± 2 nm ZnO NPs
Control
Gram negative B. subtilis S. paratyphi B K. pneumoniae
12.00 ± 1.00a 7.67 ± 0.58a 5.67 ± 0.58a
5.67 ± 0.58a 6.33 ± 0.58a -
5.33 ± 0.58a 5.67 ± 0.58a -
22 ± 0.84 18 ± 0.71 20 ± 0.71
Gram positive S. epidermidis E. aerogenes S. aureus MRSA
17.00 ± 1.00b 6.00 ± 1.00a 6.67 ± 0.58a
15.67 ± 0.58b 5.67 ± 0.58a 6.00 ± 1.00a
13.47 ± 0.58b 6.00 ± 1.00a 5.33 ± 0.58a
14 ± 0.71 22 ± 0.71 30 ± 0.90
5.67 ± 0.58a 5.00 ± 1.00a
6.00 ± 1.00a 6.00 ± 0.45a
5.33 ± 0.58a 5.00 ± 0.25a
28 ± 0.71 26 ± 0.90
fungi C. albicans M. pachydermatis
(-) no activity Statistically significant P < 0.05; P < 0.01 when compared to control Statistically insignificant when compared to control antibiotic (streptomycin – antibacterial; ketoconazole – antifungal)
a
b
plant cells at relatively higher concentration and lower particle size [12, 32]. They are not expected to be toxic at very low concentrations. In fact, it has been shown that ZnO protects against intestinal diseases by protecting intestinal cells from Escherichia coli infection by inhibiting the adhesion and internalisation of bacteria [33]. Therefore antibacterial activity of ZnO NPs from the present results suggests that ZnO NPs may play differential response to various tested microorganisms. This may be consistent with the prediction that S. epidermis can metabolise Zn2+ as an oligoelement [33]. Similarly, metal-ion homaeostasis is important for bacterial life because of their involvement in the regulation of a wide array of metabolic functions as coenzymes, cofactors and catalysts, and as structural stabilisers of enzymes and DNA-binding proteins [34]. However, excess metal or metal ions are toxic for bacterial cells. Therefore certain bacteria have developed mechanisms to regulate the influx and efflux processes to maintain the steady intracellular concentration of metal ions, including the Zn2+ ion. Zinc is essential for all organisms, because it plays a critical role in the catalytic activity and/or structural stability of many proteins [35]. Limited studies have reported the mechanisms and regulation of zinc transport in bacteria [35, 36]. Several studies suggest that bacteria appear to possess a specific energy-dependent zinc transport system [37]. The genes responsible for the transport of zinc ions have been characterised in several bacteria, including S. aureus and B. subtilis [12]. In S. aureus, ZntA and ZntR genes have been characterised, and it has been shown that ZntA, a transmembrane protein, is responsible for the efflux of zinc and cobalt ions and that ZntR encodes for a Zn-responsive regulatory protein [12, 35]. In Salmonella typhimurium, ZnuABC genes encode a high-affinity zinc uptake system and pitA gene is responsible for low-affinity system [38]. Studies have reported that ATP-binding protein ZurA is responsible for zinc uptake in S. epidermis (source: UniprotKB/TrEMBL; accession no: E6JMH7). Metal NPs breaks down the membrane permeability barrier and it may be possible that ZnO NPs perturbs the membrane lipid bilayer in case of fungal organisms [39]. Much of the differential antibacterial activity of ZnO NPs on various microorganisms will depend on cell wall integrity or membrane structures of the respective bacteria as the outer membrane structure of Gram-negative 114 & The Institution of Engineering and Technology 2014
bacteria is remarkably different from that of Gram-positive bacteria. It is also well-known that different strains within a species vary significantly in terms of infectivity, and tolerance to various agents including antibiotics [28, 40]. The higher antibacterial activity of ZnO NPs in S. epidermis may involve the production of reactive oxygen species and the deposition on the surface or accumulation in the cytoplasm of the cells as observed in earlier studies for S. aureus [40]. However, no inhibition is observed for K. pneumoniae, which requires further investigations. The highest concentration (200 µg.ml−1) and lowest particle size 15 nm has been found to be strongly inhibit the survival of pathogenic microorganisms tested. The results obtained in our study indicate that the inhibitory efficacy of ZnO NPs is very much dependent on its chosen concentration and size, which is similar to other findings [6, 32, 41]. These primary findings suggest that ZnO NP not exceeding 25 nm can be used externally to control the spreading of bacterial infections. In the prevention and control of bacterial spreading and infections, the main target is the cell wall structure. The cell wall of most pathogenic bacteria is composed of surface proteins for adhesion and colonisation, and components such as polysaccharides and teichoic acid that protect against host defenses and environmental conditions [41]. These components are charged macromolecules; therefore specific interactions to disrupt their main function and the cell membrane location may be triggered by introducing specific groups on the surface of the NPs. It has been reported that certain long-chain polycations coated onto surfaces can efficiently kill both Gram-positive and Gram-negative bacteria [42, 43]. These studies have indicated that families of unrelated hydrophobic groups are equally efficient in killing bacteria [12]. The present study suggests that ZnO NPs might have inhibited the growth of fungi by interfering with cell function and causing deformation in fungal hyphae [31]. Several studies such as electron microscopic imaging and Raman spectroscopy have reported the antifungal and antibacterial effects of ZnO NPs and their interaction by traditional microbiological plating [12, 31]. Although possible mechanisms have been proposed in earlier reports [12, 41], still the exact mechanism underlying the antimicrobial activity of the ZnO NPs remains to be understood. It can be concluded that ZnO NPs constitute an effective antimicrobial agent against common pathogenic IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
www.ietdl.org
Fig. 4 Antimicrobial activity of ZnO NPs against a B. subtilis MTCC 441 b S. paratyphi B c K. pneumoniae MTCC 109 d S. epidermidis MTCC 3615 e E. aerogenes MTCC 111 f S. aureus MRSA g C. albicans TCC 227 h M. pachydermatis Control – streptomycin (antibacterial) and ketoconazole (antifungal)
IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
115
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www.ietdl.org Table 2 MIC of ZnO NPs Organism
15 ± 4 nm ZnO NPs
25 ± 4 nm ZnO NPs
38 ± 2 nm ZnO NPs
Control
Gram negative B. subtilis S. paratyphi B K. pneumoniae S. typhimurium
50 100 200 >200
200 100 >200 >200
>200 200 >200 >200
100
Gram positive S. epidermidis E. aerogenes S. aureus MRSA
25 100 200
25 200 200
50 200 200
6.25
ISSN 1751-8741
Size-dependent antimicrobial response of zinc oxide nanoparticles Loganathan Palanikumar1, Sinna Nadar Ramasamy1, Chandrasekaran Balachandran2 1
Crystal Growth Centre, Anna University, Chennai 600025, India Division of Microbiology, Entomology Research Institute, Loyola College, Chennai 600034, India E-mail: [email protected]
2
Abstract: Antibacterial and antifungal activities of zinc oxide nanoparticles (ZnO NPs) were investigated against infectious microorganisms. ZnO NPs were prepared by wet chemical precipitation method varying the pH values. Particle size and morphology of the as-prepared ZnO powders were characterised by X-ray diffraction, Fourier transform infrared spectroscopy and transmission electron microscope. The zone of inhibition by NPs ranged from 0 to 17 mm. The lowest minimum inhibitory concentration value of NPs is 25 µg.ml−1 against Staphylococcus epidermidis. These studies demonstrate that ZnO NPs have wide range of antimicrobial activities towards various microorganisms. The results obtained in the authors’ study indicate that the inhibitory efficacy of ZnO NPs is significantly dependent on its chosen concentration and size. Significant inhibition in antibacterial response was observed for S. epidermidis when compared with control antibiotic.
1
Introduction
As particles are reduced from a micrometre to nanometer size, the resulting properties can change dramatically. For example, electrical conductivity, hardness, active surface area, chemical reactivity and biological activity are all known to be altered. Medicinal sciences are investigating the use of nanotechnology to improve medical diagnosis and treatments [1–3]. The bactericidal effectiveness of metal NPs has been suggested to be because of both particle size and high surface-to-volume ratio. Such characteristics should allow them to interact closely with bacterial membranes, rather than the effect being solely because of the release of metal ions [4]. Inorganic antibacterial agents are more stable at high temperatures and pressures compared with the organic materials, and the metallic oxide powders could be suggested as powerful antimicrobial agents [5]. Zinc oxide is traditionally used for the photocatalytic oxidation of organic and inorganic pollutants and sensitisers for the photodestruction of cancer cells, bacteria and viruses via oxidative damage [6–8]. NPs generally produces toxic effects with plasma proteins, transient inflammatory, cell injury effects leading to inflammation and fibrosis. The NPs have shown elevated blood biochemical parameters, accumulation of foamy alveolar macrophages, degenerated alveolar macrophages indicating alveolar lipoproteinosis. In humans, some types of NPs which are used for cosmetics etc. may result in skin disease. However, ZnO#ZnS quantum dots heterojunctions have been reported to enhance transportation of flavonoid glycosides in blood [7, 8]. Hence, it is appropriate to assume that ZnO is non-toxic and so it has been taken for this research study. IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
ZnO NPs are widely used in many consumer products like cosmetics, toothpaste, textiles and skin lotions [9]. Staphylococcus aureus and S. epidermidis are natural inhabitants of human and animal skin, but it can sometimes cause infections that affect many organs [10]. The pathogenic bacteria Klebsiella pneumoniae and Enterobacter aerogenes are the major causative agents of nosocomial infections [11]. Since 1980s, methicillin-resistant S. aureus (MRSA) has been commonly linked with hospital-associated infections [12]. Paratyphoid fever is caused by Salmonella enterica serotypes Paratyphi A, Paratyphi B or Paratyphi C. As per literature, an estimated 5 400 000 cases of paratyphoid fever occurred globally in 2000 [13]. Serotype Paratyphi B var. L (+) tartrate (+) causes a typical Salmonella gastroenteritis instead of enteric fever [14]. Candida albicans and Malassezia pachydermatis are the most frequent human and animal pathogens [15, 16]. The antibacterial and antifungal activities of bulk ZnO powders and ZnO NPs have been demonstrated already [12, 17]. However, little is known about the activity of ZnO NPs in the range 15–50 nm range towards the infectious microorgansims. Towards this purpose, evaluation has been made on the antibacterial and antifungal effect as a function of synthesised ZnO NPs size based on the inhibition zone in the disk diffusion tests with determining minimum inhibitory concentration (MIC).
2 2.1
Materials and methods Synthesis of ZnO NPs
ZnO NPs were synthesised with a slight modification suggested by Wu et al. [18] from aqueous solutions of zinc 111
& The Institution of Engineering and Technology 2014
www.ietdl.org nitrate [Zn (NO3)2.6H2O; purchased from Fischer Chemicals, Mumbai, India; purity 96%] and hexamethyltetramine (HMT; C6H12N4; purchased from Qualigens chemicals Ltd., Mumbai, India; purity 99%). The two chemicals were mixed separately with milli-Q water to a concentration of 0.05 M for the [Zn (NO3)2] solution, and 1.5 M for the HMT solution. The separate solutions are stirred for 30 min each, and then mixed with 130 rpm stirring. The solutions was adjusted to the desired pH (5.0, 6.0 and 7.2) and heated to 80°C for 45 min. The product is collected by centrifugation (Compufuge, Remi Electrotechnik Limited, Thane, India). The ammonium hydroxide solution (1 N) was added to pH 5.0 synthesised solutions to enhance the formation of ZnO at 80°C [19]. 2.2
Particle characterisation
Powder X-ray diffraction (XRD, Seifert, JSO-DE BYEFLEX 2002, Germany) was utilised to identify the crystalline phase composition and purity. The phase was found to be hexagonal and no impurity peaks were found. The morphology and grain size of the ZnO were observed by TECNAI G2 Model T-30 S-twin high-resolution transmission electron microscopy (HRTEM). The quality of the ZnO NPs was analysed by Fourier transform infrared spectroscopy (FTIR, Perkin Elmer Spectrum One). 2.3
Microbial organisms
The following bacteria and fungi were used for the experiment. Bacteria: Salmonella paratyphi B, K. pneumoniae MTCC 109, Bacillus subtilis MTCC 441, E. aerogenes MTCC 111, Staphylococcus epidermidis MTCC 3615, Methicillin resistant-MRSA. The reference cultures were obtained from Institute of Microbial Technology (IMTECH; Chandigarh, India 160 036); fungi: C. albicans MTCC 227 and M. pachydermatis were obtained from the Department of Microbiology (Christian Medical College, Vellore, Tamil Nadu, India). 2.4
Antimicrobial assay
Antibacterial and antifungal activities were carried out using disc-diffusion method [20, 21]. Petri plates were prepared with 20 ml of sterile Mueller Hinton agar (MHA) (Himedia, Mumbai). The test cultures were swabbed on the top of the solidified media and allowed to dry at room temperature for 10 min. The suspension of NPs (in milli-Q water) was sonicated to prepare required suspensions such as 50, 100 and 200 µg.ml−1 added to each well (diameter of 8 mm) separately. Preliminarily, a broad range of concentrations (10–200 µg.ml−1) were chosen for antimicrobial assay (data not shown). Based on those results, three concentrations were chosen for the present work as a function of size. The suspension of NPs were loaded on the wells of the medium and left for 30 min at room temperature for diffusion. Negative control was prepared using respective solvents. Streptomycin (25 µg/ disc; purchased from Himedia Chemicals Ltd., Mumbai, India) was used as positive control against bacteria. Ketoconazole (25 µg/disc; purchased from Himedia Chemicals Ltd., Mumbai, India) was used as positive control for fungi. The plates were incubated for 24 h at 37°C for bacteria and for 48 h at 28°C for fungi. Zones of inhibition were recorded in millimetres by repeating the experiment thrice for each concentration. 112 & The Institution of Engineering and Technology 2014
2.5
Minimum inhibitory concentration
MIC studies of the NPs were performed according to the standard reference methods for bacteria [22], for filamentous fungi [21] and yeasts [23]. The required concentrations (3.125, 6.25, 12.5, 25, 50, 100 and 200 µg. ml−1) of the NPs were dispersed in milli-Q water and diluted to give serial two-fold dilutions that were added to each medium in 96 well plates. A volume of 100 µl (inoculum) from each well was inoculated. The antifungal agents ketoconazole for fungi and streptomycin for bacteria were included in the assays as positive controls. For fungi, the plates were incubated for 48–72 h at 28°C and for bacteria the plates were incubated for 24 h at 37°C. The MIC for fungi was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. 5 µl of tested broth was placed on the sterile MHA plates for bacteria and incubated at respective temperatures. The MIC for bacteria was determined as the lowest concentration of the compound inhibiting the visual growth of the test cultures on the agar plate. 2.6
Statistical analysis
The differences in antimicrobial activity of ZnO NPs in comparison with control were assessed by one-way analysis of variance [24]. Dunnett’s post hoc test was employed to compare the significant difference between control and different exposure concentrations. This statistical analysis was carried out using computer-assisted software program Graph Pad Prism version 5.0. Other statistical analysis was carried out using Microsoft Office Excel 2003.
3
Results
The present study employed a low-temperature synthesis method to prepare ZnO NPs. Zinc nitrate and HMT solutions were mixed at 80°C for 45 min at different pH and a precipitation reaction occurred, after the pH was 8.2. The XRD pattern of synthesised ZnO NPs demonstrated that the ZnO is crystalline in nature, and the diffraction peaks matched very well with a hexagonal zincite (wurtzite) phase of ZnO (Fig. 1). The diffraction pattern and inter-planar spacing closely matched those in the standard diffraction pattern of ZnO (Powder diffraction file ICDD
Fig. 1 XRD pattern of ZnO NPs prepared at different pH IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
www.ietdl.org 36-1451 a = 3.249 Å and c = 5.206 Å). The XRD peaks show (100), (002), (101), (102), (110), (103), (200) and (201) reflection lines of hexagonal zincite phase of ZnO particles. No characteristic peaks of any impurities were detected suggesting good quality and widened peak were detected. The particle size based on broadening was analysed by Scherrer formula, modified form of Williamson–Hall analysis and size–strain plot method. The crystalline size can be calculated using the following equation 2 2 dhkl bhkl cosu = K/D dhkl bhkl cosu + (1/2)2 where K is the constant that depends on the shape of the particles. The particle size determined from the slope of linearly fitted data and the root of the y-intercept gives the strain [25].
The quality of the as-synthesised product without any heat treatment was analysed by the FTIR spectroscopy. FTIR confirm the formation of ZnO. Fig. 2 shows the FTIR spectrum acquired in the range of 400–4000 cm−1. The band at 535 cm−1 corresponds to the stretching vibration of Zn–O bond. The broadening corresponding to Zn–O peak at 535 cm−1 is broadened. This may be because of the nanocrystalline nature of the compound. The broad absorption bands in the range 3900–2350 and 1637 cm−1 correspond to the presence of the surface hydroxyl groups [26]. C–OH stretching (1387 cm−1) are detected from the FTIR spectrum [27]. The band at 1387 cm−1 corresponds to the CH2 deformation and absorption bands at 1235–1125 cm−1 are responsible for CN stretch. The band at 998 and 897 cm−1 corresponds to CH2 rock [28]. Although these extra peaks because of the starting material hexamethylenetetraamine appear in the as-prepared material, the tetramethyl amine is expected to go-off on heat treatment, before we go for antimicrobial and MIC studies. The morphology and size distribution of the ZnO NPs prepared at various pH are shown in Fig. 3. It can be clearly observed that the size of NPs was reduced with
Fig. 3 FTIR spectrum of as-prepared ZnO NPs (38 nm)
decreasing pH. The number of hexagonal-shaped NPs increased as the pH decreased. The TEM images confirmed the formation of hexagonal structure of ZnO and are in agreement with the XRD results. The particle size of the ZnO nanograins prepared at different pH was 38 ± 2; 25 ± 4 and 15 ± 4 nm in as-prepared (pH 7.2), pH 6.0 and pH 5.0, respectively. Modified Scherrer formula gives 32 ± 4; 25 ± 4 and 15 ± 4 nm, respectively, for the above samples. 3.1
The antibacterial and antifungal activity of ZnO NPs was compared for infectious Gram-positive and Gram-negative bacteria and fungus. The results of antimicrobial activity of different size of ZnO NPs were shown in Table 1 and Figs. 4a–h. The zone of inhibition (in mm) reflects the magnitude of susceptibility of the microorganism. The strains susceptible to NPs exhibit larger zone of inhibition, whereas resistant strains exhibit smaller or no zone of inhibition. Significant difference between control antibiotics (streptomycin and ketoconazole) were observed for all the NPs treatment, whereas insignificant zone of inhibition were observed for S. epidermis. No antimicrobial response was observed for 25 and 38 nm ZnO NPs against K. pneumonia. Table 2 presents the MICs of ZnO NPs for infectious microorganisms. Highest MIC response was observed for S. epidermis.
4
Fig. 2 TEM images of ZnO NPs prepared at different pH a As prepared b pH6.0 c pH5.0 IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
Antimicrobial and MIC studies
Discussion
The re-emergence of infectious diseases poses a serious threat to public health worldwide, and the increasing rate of the appearance of antibiotic-resistant strains in a short period of time within both Gram-positive and Gram-negative bacteria and fungal microorganisms is a major public health concern [12]. Alternative therapeutics to control and prevent the spread of infections in both community and hospital environments are required [29]. ZnO NPs are much more effective agents in controlling the growth of various microorganisms [12, 30, 31]. Our preliminary studies show that the reduced particle size had greater efficacy in inhibiting the growth of microorganisms. Similarly, metals and metal oxides such as ZnO NPs are known to be toxic to host human and 113
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www.ietdl.org Table 1 Antimicrobial activity of ZnO NPs (zone of inhibition in mm; 200 µg.ml−1) Organism
15 ± 4 nm ZnO NPs
25 ± 4 nm ZnO NPs
38 ± 2 nm ZnO NPs
Control
Gram negative B. subtilis S. paratyphi B K. pneumoniae
12.00 ± 1.00a 7.67 ± 0.58a 5.67 ± 0.58a
5.67 ± 0.58a 6.33 ± 0.58a -
5.33 ± 0.58a 5.67 ± 0.58a -
22 ± 0.84 18 ± 0.71 20 ± 0.71
Gram positive S. epidermidis E. aerogenes S. aureus MRSA
17.00 ± 1.00b 6.00 ± 1.00a 6.67 ± 0.58a
15.67 ± 0.58b 5.67 ± 0.58a 6.00 ± 1.00a
13.47 ± 0.58b 6.00 ± 1.00a 5.33 ± 0.58a
14 ± 0.71 22 ± 0.71 30 ± 0.90
5.67 ± 0.58a 5.00 ± 1.00a
6.00 ± 1.00a 6.00 ± 0.45a
5.33 ± 0.58a 5.00 ± 0.25a
28 ± 0.71 26 ± 0.90
fungi C. albicans M. pachydermatis
(-) no activity Statistically significant P < 0.05; P < 0.01 when compared to control Statistically insignificant when compared to control antibiotic (streptomycin – antibacterial; ketoconazole – antifungal)
a
b
plant cells at relatively higher concentration and lower particle size [12, 32]. They are not expected to be toxic at very low concentrations. In fact, it has been shown that ZnO protects against intestinal diseases by protecting intestinal cells from Escherichia coli infection by inhibiting the adhesion and internalisation of bacteria [33]. Therefore antibacterial activity of ZnO NPs from the present results suggests that ZnO NPs may play differential response to various tested microorganisms. This may be consistent with the prediction that S. epidermis can metabolise Zn2+ as an oligoelement [33]. Similarly, metal-ion homaeostasis is important for bacterial life because of their involvement in the regulation of a wide array of metabolic functions as coenzymes, cofactors and catalysts, and as structural stabilisers of enzymes and DNA-binding proteins [34]. However, excess metal or metal ions are toxic for bacterial cells. Therefore certain bacteria have developed mechanisms to regulate the influx and efflux processes to maintain the steady intracellular concentration of metal ions, including the Zn2+ ion. Zinc is essential for all organisms, because it plays a critical role in the catalytic activity and/or structural stability of many proteins [35]. Limited studies have reported the mechanisms and regulation of zinc transport in bacteria [35, 36]. Several studies suggest that bacteria appear to possess a specific energy-dependent zinc transport system [37]. The genes responsible for the transport of zinc ions have been characterised in several bacteria, including S. aureus and B. subtilis [12]. In S. aureus, ZntA and ZntR genes have been characterised, and it has been shown that ZntA, a transmembrane protein, is responsible for the efflux of zinc and cobalt ions and that ZntR encodes for a Zn-responsive regulatory protein [12, 35]. In Salmonella typhimurium, ZnuABC genes encode a high-affinity zinc uptake system and pitA gene is responsible for low-affinity system [38]. Studies have reported that ATP-binding protein ZurA is responsible for zinc uptake in S. epidermis (source: UniprotKB/TrEMBL; accession no: E6JMH7). Metal NPs breaks down the membrane permeability barrier and it may be possible that ZnO NPs perturbs the membrane lipid bilayer in case of fungal organisms [39]. Much of the differential antibacterial activity of ZnO NPs on various microorganisms will depend on cell wall integrity or membrane structures of the respective bacteria as the outer membrane structure of Gram-negative 114 & The Institution of Engineering and Technology 2014
bacteria is remarkably different from that of Gram-positive bacteria. It is also well-known that different strains within a species vary significantly in terms of infectivity, and tolerance to various agents including antibiotics [28, 40]. The higher antibacterial activity of ZnO NPs in S. epidermis may involve the production of reactive oxygen species and the deposition on the surface or accumulation in the cytoplasm of the cells as observed in earlier studies for S. aureus [40]. However, no inhibition is observed for K. pneumoniae, which requires further investigations. The highest concentration (200 µg.ml−1) and lowest particle size 15 nm has been found to be strongly inhibit the survival of pathogenic microorganisms tested. The results obtained in our study indicate that the inhibitory efficacy of ZnO NPs is very much dependent on its chosen concentration and size, which is similar to other findings [6, 32, 41]. These primary findings suggest that ZnO NP not exceeding 25 nm can be used externally to control the spreading of bacterial infections. In the prevention and control of bacterial spreading and infections, the main target is the cell wall structure. The cell wall of most pathogenic bacteria is composed of surface proteins for adhesion and colonisation, and components such as polysaccharides and teichoic acid that protect against host defenses and environmental conditions [41]. These components are charged macromolecules; therefore specific interactions to disrupt their main function and the cell membrane location may be triggered by introducing specific groups on the surface of the NPs. It has been reported that certain long-chain polycations coated onto surfaces can efficiently kill both Gram-positive and Gram-negative bacteria [42, 43]. These studies have indicated that families of unrelated hydrophobic groups are equally efficient in killing bacteria [12]. The present study suggests that ZnO NPs might have inhibited the growth of fungi by interfering with cell function and causing deformation in fungal hyphae [31]. Several studies such as electron microscopic imaging and Raman spectroscopy have reported the antifungal and antibacterial effects of ZnO NPs and their interaction by traditional microbiological plating [12, 31]. Although possible mechanisms have been proposed in earlier reports [12, 41], still the exact mechanism underlying the antimicrobial activity of the ZnO NPs remains to be understood. It can be concluded that ZnO NPs constitute an effective antimicrobial agent against common pathogenic IET Nanobiotechnol., 2014, Vol. 8, Iss. 2, pp. 111–117 doi: 10.1049/iet-nbt.2012.0008
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Fig. 4 Antimicrobial activity of ZnO NPs against a B. subtilis MTCC 441 b S. paratyphi B c K. pneumoniae MTCC 109 d S. epidermidis MTCC 3615 e E. aerogenes MTCC 111 f S. aureus MRSA g C. albicans TCC 227 h M. pachydermatis Control – streptomycin (antibacterial) and ketoconazole (antifungal)
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www.ietdl.org Table 2 MIC of ZnO NPs Organism
15 ± 4 nm ZnO NPs
25 ± 4 nm ZnO NPs
38 ± 2 nm ZnO NPs
Control
Gram negative B. subtilis S. paratyphi B K. pneumoniae S. typhimurium
50 100 200 >200
200 100 >200 >200
>200 200 >200 >200
100
Gram positive S. epidermidis E. aerogenes S. aureus MRSA
25 100 200
25 200 200
50 200 200
6.25
