ZnO/CdS Nanocomposite: An Anti-Microbial and Anti-Biofilm Agent ()
1. Introduction
Emergence of the antibiotics resistance in pathogens has become a serious health problem which requires an immediate attention. It is known that over 70% of bacterial infections are resistant to one or more of the antibiotics that are generally used to eradicate the infection [1]. There are in general two reasons for antimicrobial resistance; one being the misuse or overuse of antibiotics and second being the ability of microorganisms to form biofilm, a more prominent issue [2]. Biofilms are defined as conglomerations of bacterial cells and it remains with extra polymeric substance (EPS). EPS behaves as diffusion barrier and does not allow an entry of antibiotics inside the biofilm, thereby protecting cells residing inside the biofilm. The consequences of the restricted entry of antibiotics inside biofilm allow cells to grow and proliferate by drawing nutrients from biofilm, eventually emerging as multidrug resistant pathogens [3]. In the medical science, a recent survey shows that up to 60% of all human infections are owing to the biofilm. Therefore, effective strategies which will eradicate the microorganisms and biofilm have become a need of an hour [4]. In this regard, nanomaterials owing to their nanosized effect (higher surface to volume ratio) showed unique physical and chemical properties different from bulk materials which offer wide applications in various fields. Recently, metal nanoparticles have been reported for antimicrobial and antibiofilm activities [5]. However, semiconductor metal oxide nanostructures are better over all existing nanocomposites owing to very good property of photon absorption as well as efficient transport of photogenerated electron hole charge carriers [6]. Among semiconductor metal oxide, e.g. zinc oxide (ZnO) is tremendously explored due to wide band gap energy of 3.37 eV and large exciton binding energy (60 meV) [7]. Another reason for wide applications of ZnO nanoparticles in antimicrobial and antimicrobial research has been its constant photo-catalytical activities under harsh processing conditions [8]. Upon illumination, ZnO nanoparticles generate the electron hole pairs, and produce reactive oxygen species (ROS), which oxidizes organic matter and, thus, imparts biocidal property to ZnO [9]. However, ZnO nanoparticles are having a very low efficiency for the separation of electron hole pairs due to fast recombination of charge carriers [10]; therefore, efforts have been made to suppression of the recombination of photogenerated electron-hole pairs in ZnO nanoparticles. In this regard, ZnO nanoparticles have been doped or conjugated with other nanoparticles such as ZnO/CdTe for antimicrobial applications [11], ZnO/CdSe for photoelectrode for splitting water [12], ZnO/CdS for enhanced field emission behavior [13], ZnO/CdS for enhanced photocatalytic activity [14], ZnO/CdS for antibacterial activity [15], ZnO/CdS for enhanced photocatalytic H2 evolution [16], and ZnO/CdS nanocomposite for Solar cell [17]. Herein, we have attempted to enhance the photocatalytic efficiency ZnO nanoparticles by synthesizing a composite with CdS nanoparticles, and further evaluated its antimicrobial and antibiofilm.
2. Materials and Methods
2.1. Chemicals
The as-synthesized ZnO/CdS nanocomposites were synthesized by using analytical grade zinc nitrate hexahydrate (Zn(NO3)2∙6H2O, 98%), potassium hydroxide (KOH, 99%), cadmium nitrate (Cd(NO3)2), thiourea (SC(NH2)2), polyvinyl pyrrolidone (PVP), without further purifications.
2.2. Synthesis of ZnO Nanostructure
In the typical of ZnO nanostructures, 1 mmol of Zn (NO3)2∙6H2O and 5 mmol of KOH were dissolved in 100 mL aqueous solution. Then mixture immediately transferred into a Teflon-lined stainless-steel autoclave. The hydrothermal synthesis was carried out at 120˚C etc. for 2 h duration. After completion of the synthesis duration, product washed with distilled water and dried in oven at 70˚C.
2.3. Synthesis of ZnO-CdS Heterostructures
In addition to ZnO synthesis process 0.63 mM Cd(NO3)2, 1.9 mM PVP and 0.0150 g SC(NH2)2 were added. Then mixture immediately transferred into a Teflon-lined stainless-steel autoclave (200 mL). The hydrothermal synthesis was carried out at temperatures 120˚C etc. for 2 h duration. After completion of the synthesis duration, product washed with distilled water and dried in oven at 70˚C.
2.4. Antimicrobial Study
Antimicrobial study was performed by measuring growth inhibition ability of ZnO nanostructures and ZnO/CdS nanocomposite by using model organisms such as Bacillus subtilis NCIM 2063 and Escherichia coli NCIM 2931 via colony count method under 60 - 100 Lux intensity of visible light. ZnO nanostructures and ZnO/CdS nanocomposite in the concentration range of 0 to 20 μg/mL each were added to Muller Hinton broth, and each one separately inoculated with B. subtilis and E. coli (final cell density of 1 × 106 colony forming units per mililitre (CFU/mL)) and incubated at 37˚C for 15 h. After 15 h, viable numbers of cells were recorded as CFU/mL in comparison to control (without nanomaterials). The data were confirmed as survival rates (CFU/mL), (100% survival in control). Morphological alterations on the surface of microorganisms were observed under FESEM. In short, the cells of B. subtilils and E. coli were grown to mid log phase (approximately 1 × 107 cells/mL) and treated with respective MIC concentration of ZnO and ZnO/CdS nanocomposite for 3 h at 37˚C and 200 r.p.m. The cells were collected by centrifugation at 11,000 g for 15 min at 4˚C. The pellets were then washed 3 times with 0.1 M phosphate buffer of pH 7.4 and fixed in 2.5% glutaraldehyde at 4˚C for 4 h. After rinsing 2 - 3 times with buffer, the pellets were dehydrated in ethanol serials (10% - 100%, 20 min per step), and then dried in air. Finally, the images were seen under FESEM [18].
2.5. Antibiofilm Study
Antibiofilm study of ZnO and ZnO/CdS nanocomposite was studied in Pseudomonas aeruginosa at 60 - 100 Lux intensity of visible light. It was carried out by using crystal violet retention assay because crystal violet has an affinity towards polysaccharides of biofilm. In summary, the P. aeruginosa was grown overnight in Luria Bertaini (LB) medium at 37˚C with agitation. Later, the culture was diluted with LB medium (OD600—0.02), and 50 µL of the diluted culture was added to 950 µL of LB medium and allowed to form biofilm. After formation of biofilm on polystyrene plastic surfaces, the planktonic cells (cells which are suspended in the medium) were replaced with fresh medium supplemented with 0 - 125 µg/mL ZnO and ZnO/CdS nanocomposite, separately, and incubated statically for 20 h at 37˚C. After incubation, planktonic bacteria were removed, and the biofilms were washed 2 - 3 times with phosphate buffered saline buffer. Washed biofilms were fixed with 1 mL of methanol. After 15 min, the methanol was removed, and the plates were dried at room temperature. Crystal violet (0.1% in water) was then added to each well (1 mL/well), and the plates were incubated for 15 min at room temperature. Crystal violet was then removed, and stained biofilms were washed three times with 1 mL of water. Acetic acid (33% in water) was added to the stained biofilms in order to solubilize the crystal violet, and the absorbance of the solution was read at 590 nm with a spectroscopy (Schimadzu, Japan) [18].
3. Result & Discussion
3.1. X-Ray Diffraction (XRD)
A typical XRD patterns of ZnO (red coloured) & ZnO/CdS (black colored) nanocomposites (Figure 1(a) and Figure 1(b)), show a set of well-defined crystal planes are indexed to the hexagonal wurtzite phase, with lattice constants, a = 0.325 nm and b = 0.520 nm (JCPDS: 36-1451) & ZnO/CdS nanocomposite exhibits a set of well-defined crystal planes are indexed to the hexagonal wurtzite phase of ZnO (a = 0.3249 nm and c = 0.5206 nm; JCPDS File: 036-1451) and observation of the diffraction spectrum shows the occurrence of some low intensity crystal planes, which are indexed to the cubic phase of CdS (JCPDS File: 080-0019). The crystal planes correspond to the (111), (220) and (311) of cubic CdS (shown in the inset). CdS nanoparticle has a higher full width half maxima (FWHM) of the crystal planes due to smaller nanoparticle size compare to ZnO. No crystal planes corresponding to other phases or impurities were observed. Thus, the XRD results evidently show the formation of ZnO/CdS nanocomposite.
3.2. Optical Properties
The optical properties of ZnO and ZnO/CdS nanocomposite samples were studies by UV-visible diffused reflectance (UV-Vis-DRS) and photoluminescence spectroscopy (PL) at taken at room temperature. The UV-visible diffused reflectance (UV-Vis-DRS) of ZnO/CdS nanocomposite was shown in Figure 1(d). The spectrum of only ZnO nanostructure which illustrates the sharp absorption band
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Figure 1. (a) XRD of ZnO and ZnO-CdS nanocomposite; (b) (Inset) XRD of ZnO and ZnO-CdS nanocomposite, (c) UV-Vis-DRS spectra of ZnO and ZnO-CdS nanocomposite; (d) PL spectra of ZnO and ZnO-CdS nanocomposites.
at 385 nm (3.22 eV) was known to originate from the band-edge absorption and with no apparent absorption in the visible region. In case of ZnO/CdS nanocomposite, the absorption spectra in which the band edges at ~385 (3.22 eV) and 520 nm (2.38 eV) are corresponds to the ZnO and CdS, respectively (Figure 1(c)). The two band edges feature in the typical spectra clearly shows the formations of nanocomposite between the UV region band (ZnO) and visible region (CdS).
3.3. Photoluminescence Spectroscopy (PL)
In order to shows the influence of CdS nanoparticles on the electronic properties of ZnO, the PL spectra of the ZnO in the range of 350 - 650 nm at room temperature is shown in Figure 1(c). In the case of pure ZnO, a narrow UV band emission at 393 nm was observed, which can be assigned to exciton recombination, a strong blue emission at ~400 - 450 nm, and a broad green band at about 554 nm as well as a strong emission at 596 nm in the yellow region, which can be attributed to intrinsic defects in ZnO as oxygen interstitials dominates the PL spectrum [19]. The intensity of the characteristic peaks in the PL spectrum of ZnO/CdS nanocomposite was smaller than that for the ZnO. The observed decrease in the overall intensity of the PL spectrum is attributed to the decrease in the oxygen vacancies [20]. We also assume that flakes like structure on the surface would possess more defects due to the faster one-dimensional (1D) crystal growth. The PL emission band shift in the visible region ~520 nm of spectrum and could be attributed to the interaction between the two semiconductors of ZnO and CdS. The change in the wavelength can be ascribed to the change in oxygen vacancy centers around CdS. The broad emission peak centered at ~520 nm (which corresponds to the green-yellow emission) can be ascribed to the shallow defect transition of CdS nanoparticles. The decrease in the hydroxyl species on the ZnO surface is due to the presence of the CdS nanoparticles in the nanocomposite [21] [22]. The conduction band edge of CdS is above to that of the ZnO. When the visible light incident on ZnO/CdS nanocomposite the excited electron-hole pairs are generated. Owing to the explicit nature of the band alignment at the interface of ZnO/CdS nanocomposite, the generated electrons are moving to the conduction band of ZnO, at the same time as the holes with the valence band of the CdS. These alienated electrons and holes have a drastically improved recombination lifetime of electron-hole pairs, and so available for redox reactions, and may play a vital role in the inhibition of growth/biofilm by the nanocomposite, as discussed further in the text.
3.4. Morphology
The morphology of ZnO nanoparticles was studies by FESEM (Figures 2(a)-(c)). It’s well-defined flakes-like three-dimension (3D) microstructures with diameters in the range of 1 - 2 μm. A close-up view of the nanosheets-built flakes-like microstructures reveals that the nanosheets are about ~10 nm in thickness, and they alternately connect with each other to form a network-like surface of the “flakes.” Similarly, in case ZnO/CdS heterostructures (Figures 2(d)-(f)), the CdS nanoparticles were seen on the surface of flake like ZnO with
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Figure 2. FESEM micrographs of ZnO ((a)-(c)) and ZnO/CdS nanocomposite ((d)-(f)).
average size ~15 - 20 nm. Further the morphological and structural analysis of ZnO and ZnO/CdS was carried out using TEM and HRTEM (Figure 2 and Figure 3). Flakes like ZnO microstructure were observed under TEM, confirming the hierarchical 3D structure with diameter around 1 - 2 μm, constructed by numerous nanosheets which have uniform thickness of ~10 nm (Figures 2(a)-(c)) and HRTEM image shows typical well defined lattice fringes width 0.26 nm which corresponding to the growth orientation along (002) plane (Figures 3(a)-(c)). Similarly, in case of ZnO/CdS nanocomposite, TEM images show that the of CdS nanoparticles on the flakes of ZnO (Figures 2(d)-(f)) (a magnified image). Magnified view shows a single crystalline nature of ZnO and CdS which is marked by with red color circles. The average size of CdS nanoparticles were about ~8 - 10 nm. The appearance of rings along with spots in the SAED pattern of the ZnO/CdS may be due to orientation disorder between the ZnO nanoflakes and the CdS nanoparticles (Figure 3(f)).
3.5. Antibacterial
The effect of different concentrations of ZnO and ZnO/CdS nanocomposites on the growth of B. subtilis and E. coli was shown in Figure 4. When the cells of the model organisms were subjected to increasing concentrations of ZnO and ZnO/CdS nanocomposite, the viability of cells decreased. The initial number of
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Figure 3. HRTEM micrographs of ZnO ((a) and (b)) and ZnO/CdS ((d)-(f)).
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Figure 4. Antibacterial activities of ZnO and ZnO/CdS on B. subtilis and E. coli (upper panel) and SEM micrographs of B. subtilis (left) and E. coli (right) in presence of ZnO/CdS.
viable number bacterial cells, approximately 6 Log CFU/mL increased to approximately 9 Log CFU/mL at 0 µg/mL ZnO and ZnO/CdS nanocomposite after 18 hrs of incubation. This represents a 100% growth (Figure 4). Upon increasing concentration of ZnO and ZnO/CdS nanocomposites, it was observed that whereas ZnO had a little effect on the growth of microorganisms, ZnO/CdS has decreased the growth by 1 to 3 Log CFU/mL. The concentration of ZnO/CdS nanocomposites required to reduce 3 Log CFU/mL in B. subtilis and E. coli was 20 μg/mL. It is interesting to note that the ZnO/CdS nanocomposites have a dissimilatory action on B. subtilis and E. coli, it is more prominent on B. subtilis, due to difference in the cellular structures. E. coli possesses an extra outermost layer made up of lipopolysaccharides, therefore, acts as a permeable barrier and restricted the damages cause to the cytoplasmic membrane. Our findings agreed with previous reports [23] [24]. The other reason for the predominance action of ZnO/CdS nanocomposite on E. coli is due the polarity of cell membrane of E. coli. Microorganism E. coli has fewer negative charges than B. subtilis, and therefore allows greater penetration of free radical such as hydroxyl radicals (•OH), superoxide peroxide anions (O•− 2) in B. subtilis and a preferred action on B. subtilis [23].
Several suggestions have been proposed to explain the mechanism of antimicrobial action of ZnO nanocomposites. The release of Zn2+ has been suggested as one of the reasons for the antimicrobial activity [24]. According to this theory, the toxicity of ZnO nanocomposite arose due to the dissolution of Zn2+ into the medium containing microorganisms. However, in our study, the Zn2+ leached into the medium were too less to inhibit the growth of microorganisms. Hence, we rule out this hypothesis. Oxidative stress mediated antimicrobial mechanisms of ZnO nanocomposites have also being suggested. According to this theory, the inactivation of bacteria by ZnO nanocomposites involves ZnO-mediated change in the permeability of membrane allowing the internalization of nanocomposites and induces the oxidative stress. However, when we assayed the molecular marker enzymes of oxidative stress viz., Catalase and Superoxide dismutase, we found that the expression levels of these two enzymes were like control. Secondly, the pore size in the cells wall of bacteria under study were small, approximately, 4 to 10 nm, so it was not practical possible to ZnO/CdS nanocomposite to enter the cell through cell membrane [25].
When the nanostructures interact with the cellular membrane of microorganisms in presence of sunlight, the electron and hole generated in which hole remains in the valence band and electron move towards the conduction band. Hence, while, electrons in the conduction band shows higher reducing power, where holes in the valence band shown higher oxidizing power [26]. As a result, when molecular oxygen reacts with electron, O•− 2 is produced in the conduction band through a reductive process. In the valence band, the hole abstracted electrons from the water and/or hydroxyl ions to generate •OH through an oxidative process. Singlet oxygen (1O2) was mostly produced indirectly from aqueous reactions of O•− 2. Whereas •OH is a strong and nonselective oxidant, it oxidizes the lipid bilayer of membrane and eventually ruptures the cell surfaces. The 1O2 was the main mediator of phototoxicity and irreversibly damaged the treated cells, causing the cell membrane oxidation and degradation. Thus, the growth inhibition in B. subtilis and E. coli occurred owing to the direct action of the ZnO/CdS nanocomposites on the cell surfaces. When we observed the cellular morphology of B. subtilis and E. coli under FESEM, we observed the surface perturbation, in agreement with the previous reports [27]. The ZnO/CdS nanostructures act on microbial cells by generating ROS on cellular membrane, which virtually oxidizes every cellular component and ruptured the cell wall. Therefore, when observing cellular membrane by SEM, we observed irregular shaped cells with pits and ruptured appearances on surfaces (Figure 4). The surface perturbation in B. subtilis and E. coli agreed with the previous reports [28].
3.6. Anti-Biofilm
Biofilm is a severe problem in biomedical sector; the medical implants and indwelling devices are severely affected due to bacterial colonization and biofilm formation. The conventional antibiotic therapies have become ineffective against device-associated biofilm [29]. Thus, to overcome the inherent resistance of biofilms to antimicrobial agents, we explored ZnO/CdS nanocomposite on the biofilm of P. aeruginosa. The most distinguishing phenotype of the biofilm mode of growth is its intrinsic resistance to antimicrobial treatment and immune response killing [30]. The cells of P. aeruginosa in presence of nutrient components of media tend to grow and form biofilm after 36 - 40 h. We assayed the biofilm by measuring the amount of crystal violet retained by biofilm, because crystal violet has an affinity towards the biofilm. Biofilm retains 100% crystal violet at 0 g/mL, however, biofilm retention was reduced to less than 35% at 50 μg/mL of ZnO/CdS nanocomposites. The formation of biofilm decreased with increase in the concentrations of ZnO/CdS nanocomposite. The disruption of biofilm is liable to extend the antimicrobial action of ZnO/CdS nanocomposite to the cells protected inside biofilm. Therefore, in order to know the viability of these cells inside biofilm, viable count of cells inside biofilm was performed. As can be seen from Figure 5, the cell viability inside biofilm after exposure
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Figure 5. Antibiofilm activity of ZnO and ZnO/CdS on P. aeruginosa (upper panel). SEM of biofilm of P. aeruginosa.
ZnO/CdS nanocomposite decreased. At 0 g/mL, cells viability was 100%, however, viability decreased to 40% at 125 μg/mL of ZnO/CdS nanocomposites. The antibiofilm activity in P. aeruginosa was also supported by topographical observation of biofilm under FESEM at 50 μg/mL ZnO/CdS nanocomposite (Figure 5). Under FESEM, the biofilm of P. aeruginosahas been reported as a smooth layer of matrix with an under-covered uniform cell i.e. cells embedded in the polysaccharide’s matrix [18], a typical of biofilm structure. However, at 50 μg/mL ZnO/CdS nanocomposite, a truncated biofilm was observed, with cells exposed to outer surfaces (Figure 5). The antibiofilm study is significant because the cells in the biofilm are not viable, and therefore, will not form biofilm. Earlier reports co-relate antibiofilm activity of nanomaterials due to anti-quorum sensing (anti-QS) sensing activity [31] [32]. In present study we rule out the QS mediated antibiofilm activity because our nanostructures also have brought about a reduction in the number of viable cells. We do not rule out the ROS mediated antibiofilm activity in P. aeruginosa because ROS species have been reported to carry out the oxidation of polysaccharides, proteins, and lipids biomolecules.
4. Conclusion
ZnO/CdS nanocomposites are promising applicant for the photocatalytic demolition of bacterial cells. ZnO/CdS nanocomposites are effective alternative to organic based drugs. Structural, optical and morphological data confirm the successful synthesis of ZnO/CdS nanocomposite. ZnO/CdS nanocomposites have shown antimicrobial activity against B. subtilis and E. coli, and the enhancement in antibacterial property by ZnO/CdS nanocomposite was concentration dependent. The ruptured appearances on bacterial cell surfaces indicate cellular membrane damage by ZnO/CdS nanocomposites. The decline in the development of biofilm in P. aeruginosa in the presence of ZnO/CdS nanocomposite shows that it is one of the excellent materials for inhibiting biofilm formations in other clinically pathogenic biofilm forming organisms. ZnO/CdS nanocomposites not only showed antibiofilm activity in P. aeruginosa, but it also inhibited the microbial population inside the biofilm and eliminates the biofilm forming ability of the microorganisms.
Acknowledgements
Rajendra Patil acknowledges support from Department Research Grants of Savitribai Phule Pune University, Pune University.